Abundant DNA 6mA methylation during early embryogenesis of zebrafish and pig

Abstract

DNA N⁶-methyldeoxyadenosine (6mA) is a well-known prokaryotic DNA modification that has been shown to exist and play epigenetic roles in eukaryotic DNA. Here we report that 6mA accumulates up to ∼0.1-0.2% of total deoxyadenosine during early embryogenesis of vertebrates, but diminishes to the background level with the progression of the embryo development. During this process a large fraction of 6mAs locate in repetitive regions of the genome.

Figure 1. 6mA modification in gDNAs of gametes and selected early embryos in both zebrafish and pig

(a,c) Quantification of 6mA modification in isolated gDNA from sperm, oocyte and various embryo stages of zebrafish (a) and pig (c) by UHPLC–QQQ–MS/MS. The mole ratios of 6mA/A are shown, error bars indicate mean±s.d. (n=3). (b,d) Immunofluorescence images of selected embryo stages of zebrafish (b) and pig (d) at single-cell level stained by anti-6mA antibody (green, rabbit polyclonal from SYSY) and anti-Histone 3 (H3) antibody (red, mouse monoclonal from Biodragon Immunotechnologies). Early embryo stages show strong fluorescence indicative of the high abundance of 6mA in gDNA, whereas the signal is diminished with the progression of the embryo development. Full embryo images are presented in Supplementary Fig. 3.

Figure 2. DNA 6mA-IP-seq reveals the distribution of 6mA across the genome of zebrafish at selected embryonic stages.

(a) Pie chart showing the distribution of 6mA peaks at 64-cell (64C) stage. The peaks located in REs are classified into subgroups based on RepeatMasker annotation. (b) 6mA peak classification for samples of 64C, 11 hpf, 12 hpf and 13 hpf. The genome background distribution is shown for comparison. The peaks are classified with the same criteria shown in a. (c) Sequence motifs of 6mA peaks in simple repeat, other REs apart from simple repeats (other RE) and non-repetitive regions (non-RE) for the 64C stage sample. Motifs were searched and generated by Homer software. The P-values for simple repeat, RE and non-RE motifs are 5e−1163, 1e−1557 and 1e−390, respectively.