Comparative Mapping of N6-Methyladenine, C5-Methylcytosine, and C5-Hydroxymethylcytosine in a Single Species Reveals Constitutive, Somatic- and Germline-Specific, and Age-Related Genomic Context Distributions and Biological Functions.

Background/objectives: The DNA methylome allows environmental signals to be converted into stable and adaptive changes in gene expression. While 5-methylcytosine (5mC) has been extensively studied, alternative epigenetic marks such as N6-methyladenine (6mA) and 5-hydroxymethylcytosine (5hmC) remain poorly understood. Comparative studies of these marks are rare, and their results are often confounded by phylogeny, tissue type, developmental stage, or methodology. Here, we aimed to disentangle the constitutive, somatic- and germline-specific, and/or age-related patterns displayed by 6mA, 5mC, and 5hmC within a single species.

Methods: We generated long-read nanopore sequencing data for somatic tissues of buff-tailed bumblebee (Bombus terrestris) males and their sperm, enabling simultaneous detection of 6mA, 5mC, and 5hmC. We used a stepwise approach to successively identify (i) constitutive patterns conserved between somatic tissues and sperm, (ii) differences between the soma and the germline, and (iii) age-related changes between young and old males.

Results: We found distinct constitutive, somatic and sperm, and age-related specific signatures in the genomic contexts, maintenance fidelity, and biological functions associated with 6mA, 5mC, and 5hmC. Sperm cells consistently displayed lower methylation entropy than did somatic tissues, indicating more stable methylation patterns in the germline. 5mC exhibited the greatest variation across all genomic contexts; 6mA and 5hmC displayed less dramatic differences. The influence of age was subtler but revealed context-dependent remodeling of methylation, particularly for 5hmC.

Conclusions: We observed that 6mA, 5mC, and 5hmC displayed constitutive, somatic- and sperm-specific, and age-related differences that were associated with distinct genomic contexts and biological functions, supporting the complementarity of these methylation marks and their diverging epigenetic roles.