Dynamic changes of DNA modifications: 5-hydroxymethyluracil and N6-methyladenine in the Drosophila melanogaster life cycle and in response to environmental factors

In this study, the level of DNA modifications was investigated in three development stages of Drosophila melanogaster (larvae, pupae, imago) and in an in vitro model (Schneider 2 cells). The analysis was carried out using the two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. Our method made it possible for the first time to analyze a broad spectrum of DNA modifications in the three stages of the Drosophila . Each stage was characterized by a specific modification pattern and the level of these compounds fluctuated throughout the D. melanogaster life cycle. The level of the DNA modification was also compared between insects bred at 25°C (optimal temperature) and at 18°C, the conclusion being that both groups differed significantly. The profound changes of N6-methyladenine and 5-hydroxymethyluracil levels during the Drosophila life cycle and as a result of bred temperature changes indicate that these DNA modifications can play important regulatory roles in response to environmental changes and/or biological conditions. Moreover, the supplementation of Schneider 2 cells with 1mM L-ascorbic acid caused a time-dependent increase in the level of 5-(hydroxymethyl)-2’-deoxyuridine. These data suggest that a certain pool of this compound may arise from the enzymatic activity of dTET protein.