A comprehensive epigenomic analysis of phenotypically distinguishable, genetically identical female and male Daphnia pulex

Abstract

Background: Daphnia species reproduce by cyclic parthenogenesis involving both sexual and asexual reproduction. The sex of the offspring is environmentally determined and mediated via endocrine signalling by the mother. Interestingly, male and female Daphnia can be genetically identical, yet display large differences in behaviour, morphology, lifespan and metabolic activity. Our goal was to integrate multiple omics datasets, including gene expression, splicing, histone modification and DNA methylation data generated from genetically identical female and male Daphnia pulex under controlled laboratory settings with the aim of achieving a better understanding of the underlying epigenetic factors that may contribute to the phenotypic differences observed between the two genders. Results: In this study we demonstrate that gene expression level is positively correlated with increased DNA methylation, and histone H3 trimethylation at lysine 4 (H3K4me3) at predicted promoter regions. Conversely, elevated histone H3 trimethylation at lysine 27 (H3K27me3), distributed across the entire transcript length, is negatively correlated with gene expression level. Interestingly, male Daphnia are dominated with epigenetic modifications that globally promote elevated gene expression, while female Daphnia are dominated with epigenetic modifications that reduce gene expression globally. For examples, CpG methylation (positively correlated with gene expression level) is significantly higher in almost all differentially methylated sites in male compared to female Daphnia. Furthermore, H3K4me3 modifications are higher in male compared to female Daphnia in more than 3/4 of the differentially regulated promoters. On the other hand, H3K27me3 is higher in female compared to male Daphnia in more than 5/6 of differentially modified sites. However, both sexes demonstrate roughly equal number of genes that are up-regulated in one gender compared to the other sex. Since, gene expression analyses typically assume that most genes are expressed at equal level among samples and different conditions, and thus cannot detect global changes affecting most genes. Conclusions: The epigenetic differences between male and female in Daphnia pulex are vast and dominated by changes that promote elevated gene expression in male Daphnia. Furthermore, the differences observed in both gene expression changes and epigenetic modifications between the genders relate to pathways that are physiologically relevant to the observed phenotypic differences.

DNA methylation differs extensively between strains of the same geographical origin and changes with age in Daphnia magna

“…We report an average CpG methylation of 1.2–3%, which is higher than our previous estimate for strain C32 of 0.7% [ 59 ]. This earlier estimate is more in line with those reported for other strains of D. magna at 0.25–0.85% [ 8 , 9 , 55 – 57 ]. The current higher estimate may result from incomplete bisulphite conversion during library preparation, as the percentage was elevated when filtering out all methylated CHG/CHH sites (the ‘No-CH’ dataset) which occur negligibly in D. magna [ 56 ].…”

“…Methylated CpGs were concentrated in gene bodies with a preference for exons 2 to 5 (Fig. 2 d), adding to the evidence that methylation is a directed process in Daphnia [ 8 , 9 ]. We report an average CpG methylation of 1.2–3%, which is higher than our previous estimate for strain C32 of 0.7% [ 59 ].…”

“…DNA methylation is enriched in exon sequences of various arthropods, including members of holometabolous insect orders Lepidoptera and Hymenoptera [ 4 – 7 ] and species of the Crustacea genus Daphnia [ 8 , 9 ]. Exon-enriched DNA methylation has been shown to occur in genes expressed across multiple tissues [ 6 , 7 , 10 , 11 ].…”

“…D. magna has more recently emerged as an epigenetic model organism. A variety of life-history and ecotoxicological stimuli have been shown to modify methylated cytosines (CpGs), histones and small RNA expression [ 1 , 3 , 9 , 53 – 60 ]. In this role, Daphnia species have an advantage over the model invertebrate Drosophila melanogaster .…”

“…D. melanogaster has negligible levels of adult DNA methylation and lacks the essential DNA methyltransferase genes (DNMT1 and DNMT3A/B), although this is subject to continuing debate (discussed in [ 61 ]). D. magna encodes a full complement of DNMTs and gene-body DNA methylation has been shown to correlate with increased gene expression in male versus female D. pulex [ 9 ]. This is despite low levels of overall cytosine (CpG) methylation of 0.25–0.8% estimated in D. magna [ 8 , 53 – 59 ], which is sparsely distributed across Daphnia genomes [ 8 , 9 , 53 , 55 , 56 , 58 , 59 ].…”