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.
Highlights
Daphnids are freshwater crustaceans that, dependent on environmental conditions, can reproduce either sexually or parthenogenetically
The presence of cytosine methylation and hydroxymethylation in D. pulex genomic DNA was presence cytosine methylation and using hydroxymethylation in D. pulex genomic first first The assessed by ofimmunological reaction, antibodies specific for either assessed by immunological reaction, using antibodies specific for either
Immuno-dot blot using antibody specific for 5-mC showed a very weak signal only at a dot blot using antibody specific forthe showed a very(Figure weak signal at a individuals dot corresponding to was the corresponding to the DNA of oldest individuals
Summary
Daphnids are freshwater crustaceans that, dependent on environmental conditions, can reproduce either sexually or parthenogenetically. Water flea Daphnia pulex propagates by cyclical parthenogenesis producing subitaneous eggs. With deterioration of environmental quality, usually at the end of the growing season, these animals initiate sexual reproduction which results in two diapausing eggs encased in ephippium, a protective structure modified from carapace [1,2]. Emergence from diapausing eggs in daphnids takes place in the early season during a relatively short period [3,4,5], these eggs, being resistant to external factors, can remain viable for extended time periods [6]. D. pulex exhibit different polyphenisms [7,8]. Since clonal lines are genetically identical but
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