Rearrangement of 5-carboxylcytosine patterns initiates genome reprogramming during spermiogenesis

Chromatin of male and female gametes undergoes a number of reprogramming events during a transition from germ cell to embryonic developmental program in the zygote. This process involves reorganisation of the patterns of 5-methylcytosine (5mC), a DNA modification associated with regulation of gene activity. Notably, both maternal and paternal genomes undergo Tet3-dependent oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in the one-cell embryos. Despite the exact biological functions of these oxidised forms of 5mC remain elusive, they may play specific roles in active demethylation and transcriptional regulation. Here we present the results of genome-scale analysis of 5mC/5hmC/5caC distribution in round spermatids and spermatozoa and demonstrate that reprogramming of the paternal genome begins during spermatid maturation. We show that patterns of 5caC genomic distribution are highly dynamic in spermiogenesis. Thus, whereas 5caC is eliminated from LINE1 retroposons and transcriptionally active spermiogenesis-specific genes during spermatid maturation, it is simultaneously accumulated at promoter regions and introns of the genes involved in embryo development. Surprisingly, the elimination of 5caC from LINE1 elements is not associated with their demethylation. Moreover, the genomic regions enriched in 5caC in spermatozoa do not correspond to the loci reported to experience demethylation in the zygote. Our results suggest that embryonic patterns of DNA methylation are prearranged during spermiogenesis and imply a role for 5caC in transcriptional regulation.