Tet proteins, which are known for converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), have been shown to also generate 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) from 5mC through enzymatic activity. This study demonstrates that Tet proteins can produce these new cytosine derivatives in genomic DNA of mouse embryonic stem cells and organs. The presence of 5fC and 5caC in genomic DNA was confirmed using restriction enzymes and two-dimensional thin-layer chromatography (2D-TLC). The levels of these derivatives can be increased or decreased by overexpression or depletion of Tet proteins, indicating their direct involvement in DNA demethylation. The study suggests that DNA demethylation may occur through a process involving Tet-catalyzed oxidation followed by decarboxylation. The findings also show that Tet proteins can act on 5hmC and 5fC-containing DNA substrates, further supporting the idea of iterative oxidation. The presence of 5fC and 5caC in genomic DNA was confirmed in mouse ES cells and organs, with 5caC being more reliably detected in ES cells. The study also highlights the potential for Tet proteins to work in conjunction with other DNA demethylation pathways, such as the base excision repair (BER) pathway, until the enzyme responsible for decarboxylating 5caC is identified. Overall, the study provides evidence that Tet proteins not only convert 5mC to 5hmC but also to 5fC and 5caC, expanding our understanding of DNA demethylation mechanisms.Tet proteins, which are known for converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), have been shown to also generate 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) from 5mC through enzymatic activity. This study demonstrates that Tet proteins can produce these new cytosine derivatives in genomic DNA of mouse embryonic stem cells and organs. The presence of 5fC and 5caC in genomic DNA was confirmed using restriction enzymes and two-dimensional thin-layer chromatography (2D-TLC). The levels of these derivatives can be increased or decreased by overexpression or depletion of Tet proteins, indicating their direct involvement in DNA demethylation. The study suggests that DNA demethylation may occur through a process involving Tet-catalyzed oxidation followed by decarboxylation. The findings also show that Tet proteins can act on 5hmC and 5fC-containing DNA substrates, further supporting the idea of iterative oxidation. The presence of 5fC and 5caC in genomic DNA was confirmed in mouse ES cells and organs, with 5caC being more reliably detected in ES cells. The study also highlights the potential for Tet proteins to work in conjunction with other DNA demethylation pathways, such as the base excision repair (BER) pathway, until the enzyme responsible for decarboxylating 5caC is identified. Overall, the study provides evidence that Tet proteins not only convert 5mC to 5hmC but also to 5fC and 5caC, expanding our understanding of DNA demethylation mechanisms.