About 2-6% of all cytosine residues and 70-80% of cytosine residues in CG dinucleotides in mammalian cells are methylated at the 5 position of the pyrimidine ring. The cytosine residues are methylated by DNA methyltransferases after DNA replication and can be demethylated by passive dilution during subsequent replication or by active modification of the 5-methylcytosine base. Cytosine demethylation is developmentally regulated: one wave of demethylation occurs in primordial germ cells and one wave occurs by active demethylation in the male pronucleus after fertilization.
Some mechanisms of active demethylation remain controversial, however progressive oxidation of the methyl group of 5-methylcytosine followed by base excision by thymine DNA glycosylase (TDG) has been reproducibly demonstrated in vivo (reviewed in Wu and Zhang 2011, Franchini et al 2012, Cadet and Wagner 2013, Kohli and Zhang 2013, Ponnaluri et al. 2013, Rasmussen and Helin 2016). Ten-eleven translocation proteins TET1, TET2, and TET3 are dioxygenases that first oxidize 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) (Tahiliani et al. 2009, Ito et al. 2010), which is found in significant quantities and specific genomic locations in stem cells and neurons (Kinney and Pradhan 2013). TET proteins can further oxidize 5-hmC to 5-formylcytosine (5-fC) and then 5-carboxylcytosine (5-caC) (He et al. 2011, Ito et al. 2011). G:5-fC and G:5-caC base pairs are recognized by TDG, which excises the 5-fC or 5-caC and leaves an abasic site.
TET1 in mouse is expressed in neurons and its expression depends on neuronal activity (Guo et al. 2011, Kaas et al. 2013, Zhang et al. 2013). TET1 is also found in embryonic stem cells (Ficz et al. 2011, Koh et al. 2011, Wu et al. 2011) and in primordial germ cells of mice, where it plays a role in erasure of imprinting (Yamaguchi et al. 2013). TET3 is expressed in oocytes and zygotes of mice and is required for demethylation in the male pronucleus (Gu et al. 2011, Iqbal et al. 2011). TET2 is the most highly expressed TET family protein in hemopoietic stem cells and appears to act as a tumor suppressor. TET2 is also expressed in embryonic stem cells (Koh et al. 2011).