GSE124427
A comprehensive epigenomic analysis of phenotypically distinguishable, genetically identical female and male Daphnia pulex
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. Overall design: Genomic DNA and RNA were extracted from the samples using MasterPure DNA puri?cation kit (Epicentre, USA) and RNeasy Micro Kit (Qiagen Ltd, UK), respectively as described by (Athanasio et al. 2016, 2018). EpiGenome Methyl-Seq kit (Epicentre, USA) was used to prepare the WGBS libraries and sequenced (2x80bp) using Illumina NextSeq 500 platform at the Centre for Genomics and Bioinformatics, Indiana University. The RNA-seq libraries were prepared using the Illumina TruSeq standard mRNA kit and sequenced (1x85bp) using Illumina NextSeq 500 platform at the Centre for Genomics and Bioinformatics, Indiana University. The chromatin immunoprecipitation sequencing libraries (ChIP-seq) were prepared using the iDeal-seq kit, H3K4me3 (C15410003-50, 1 ug/reaction), H3K27me3 (C15410195, 1 µg/reaction) antibodies and sequenced using Illumina HiSeq 2500 (1x50 bp) as part of a service provided by Diagenode (Belgium). Daphnia samples (30 mg wet tissue per sample) were homogenised in 1 ml of PBS/1%formaldehyde using Dounce homogenizer. The collected cells were lysed and the nuclei were collected and sonicated to a final size of 80-400 bp. The mentioned antibodies were used to prepare test samples according to the manual for the iDeal ChIP-seq kit. The IP samples and input samples were quantified using the Qubit dsDNA HS kit. Library preparation was performed on the IP and input samples using the MicroPLEX library preparation protocol on 500 pg of DNA. The amplified libraries (13 PCR cycles) were purified using AMPure beads, quantified using the Qubit ds DNA HS kit and analysed on Bioanalyzer. The prepared libraries were then sequenced on HiSeq 2500.