Epigenetic signatures in the mammalian genome, including DNA and histone modifications, control tissue- and context-specific gene expression, thereby regulating cellular differentiation and organism development (Ashe et al., 2021; Egger et al., 2004). In particular, methylation and hydroxymethylation marks on the cytosine at CpG (cytosine followed by guanine) dinucleotides tightly regulate transcription during various physiological and pathological contexts (Jin and Liu, 2018; Kriukienė et al., 2012). Methylation at the 5th carbon position of cytosine forms 5-methylcytosine (5mC), which is a repressive, heritable epigenetic mark that negatively regulates gene expression by either restricting the binding of transcription factors or favoring a non-permissive chromatin architecture by interacting with chromatin remodelers, such as methyl-DNA-binding proteins (MBDs) (Boyes and Bird, 1991; Zhu et al., 2016). The 5mC will be oxidized to form 5-hydroxymethylcytosine (5hmC), which is a transient, yet stable, epigenetic mark associated with permissive chromatin state and enhanced gene transcription, achieved either by increasing chromatin accessibility to transcription machinery or inhibiting transcriptional repressor binding proteins, such as MBD1 or methyl cytosine binding proteins 1 and 2 (MeCP1 and 2) (Branco et al., 2012).

It is estimated that the mammalian genome contains ∼3 × 107 residues of 5mC, mainly within CpG dinucleotides (Walsh and Bestor, 1999). 5mC plays a vital role in genomic stability by silencing transposable elements, allele-specific expression of imprinted genes, and X-chromosome inactivation (Schübeler, 2015). The formation of 5mC is catalyzed by a family of highly conserved enzymes known as DNA methyltransferases (DNMTs), which covalently transfer a methyl group from S-adenosyl methionine to the 5th carbon of cytosine (Edwards et al., 2017). There are three classes of DNMTs: DNMT1, DNMT2, and DNMT3A/3B/3L. Of these, DNMT1 is primarily responsible for maintenance methylation, ensuring that methylation patterns are copied during DNA replication (Goyal et al., 2006). The exact biological functions of DNMT2 remain to be characterized, although some studies show that DNMT2 regulates centromere function and tRNA modifications (Goll et al., 2006; Jeltsch et al., 2017). In contrast, DNMT3A/3B/3L are involved in de novo DNA methylation, establishing new methylation patterns during gametogenesis, embryonic development, and cell differentiation (Okano et al., 1999). While the functional significance of differential DNA methylation patterns was thoroughly investigated in cancer pathophysiology, genome-wide changes in DNA methylome are also evident during aging and in the onset and progression of several age-related diseases (Kulis and Esteller, 2010; Noroozi et al., 2021).

Despite the chemical and genetic stability of 5mC, it can still be reverted to its unmethylated cytosine through two primary pathways: passive DNA demethylation, which occurs during DNA replication when methylation marks are not preserved, and active DNA demethylation, which involves enzymatic mechanisms that directly modify or remove the methyl group (Bhutani et al., 2011). During the active DNA demethylation process, the conversion of 5mC to 5hmC is mediated by the Fe(II)- and α-ketoglutarate-dependent DNA dioxygenase family of enzymes, the ten-eleven translocases (TETs): TET1, TET2, and TET3. These enzymes oxidize 5mC to 5hmC, which can be further oxidized to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) (Tahiliani et al., 2009; Wu and Zhang, 2017). Among these oxidized cytosine derivatives, 5hmc serves as a stable and independent epigenetic mark, especially abundant in the mammalian brain (Jin et al., 2011). Dysregulated genome-wide hydroxymethylome patterns are associated with the development of several neuronal and non-neuronal diseases (Chen et al., 2016; Stöger et al., 2017). Given their influence on context and tissue-specific gene expression, 5mC and 5hmC are frequently referred to as the fifth and sixth bases of DNA (Kumar et al., 2018; Shi et al., 2017). These modifications regulate numerous biological processes, including learning, memory, aging, disease susceptibility, and overall health span (Moen et al., 2015). Therefore, precise control of 5mC and 5hmC dynamics is essential not only for tissue-specific transcriptional regulation but also for the adaptation to various forms of physiological and pathological stress throughout life.