Analysis of DNA Methylation and Hydroxymethylation in the Genome of Crustacean Daphnia pulex

Abstract

The aim of our study was to analyze the presence of 5-methyl-cytosine (5-mC) and 5-hydroxymethyl-cytosine (5-hmC) in the genome of crustacean Daphnia pulex. First, the presence of 5-mC and 5-hmC in genomic DNA was demonstrated by using antibodies specific to either 5-mC or 5-hmC. Then, analysis of 5-mC and 5-hmC using pairs of restriction enzymes with different sensitivity to methylation and hydroxymethylation confirmed the presence of both modifications in selected regions of three genes (Cox4, Cand2 and Ephx1). To get a detailed picture of 5-hmC distribution over the D. pulex genome, we performed 5-hmC enrichment and sequenced the enriched fraction using next generation sequencing and non-enriched library (input) as a control. Comparison of input and enriched libraries showed that 5-hmC in exons is twice as frequent as in introns. Functional analysis indicated that 5-hmC abundance is associated with genes that are involved in the adenylate cyclase-activating G-protein-coupled receptor signaling pathway, molting cycles, morphogenesis and cell fate determination. Genes that lack 5-hmC tend to be involved in the regulation of the transforming growth factor beta receptor signaling pathway and in many mRNA-related processes. Our results suggest that epigenetic modifications are present in the genome of D. pulex and most likely are involved in the regulation of gene expression of this crustacean.

DNA Methylation Inhibits the Expression of CFSH in Mud Crab

“…In D. magna, DNA methylation levels can be affected by Zn exposure and entailed at different histories (23,24). In D. pulex, the 5-methyl-cytosine (5-mC) and 5-hydroxymethyl-cytosine (5-hmC) can inhibit the expression of the cullin-associated NEDD8-dissociated 2 (Cand2) gene, cytochrome C oxidase subunit IV (Cox4) gene, and juvenile hormone epoxide hydrolase 1 (Ephx1) gene (25).…”

Differential gene transcription across the life cycle in Daphnia magna using a new all genome custom-made microarray

“…Homeostasis is considered important to cells on high metabolic rates in which accumulation of some metabolites can become toxic. The enrichment in methylation-related transcripts may be linked to epigenetic processes, which are common during embryo development in Daphnia and other arthropods [ 2 , 28 , 45 ]. Interestingly the cluster of genes from E1 coming from a maternal origin (E1F3) were enriched with 829 GO terms, 50% of them also present in E1 (Tables 1 and 2 ).…”

Repurposing enzymatic transferase reactions for targeted labeling and analysis of DNA and RNA

“…In particular, the C5-MTase-activated 5-hydroxymethylcytosine (hmC) residues in DNA can undergo derivatization with aliphatic thiols yielding corresponding 5-alkylthiomethyl derivatives ( Figure 1A Bottom and 4A Bottom) [48,49]. M.SssI-directed tagging of hmC with cysteamine and subsequent amine-selective biotin labeling was exploited for selective covalent capture of 5-hydroxymethylated-CG sites [48] (commercialized as EpiJET 5hmC Enrichment Kit by Thermo Fisher Scientific) and found laboratory applications [50,51]. Similar M.HhaI-directed labeling has been used for a proofof-principle demonstration of a photo-electrochemical biosensor for hmC quantitation [52].…”