Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA

Abstract

The prevalent DNA modification in higher organisms is the methylation of cytosine to 5-methylcytosine (5mC), which is partially converted to 5-hydroxymethylcytosine (5hmC) by the Tet (ten eleven translocation) family of dioxygenases. Despite their importance in epigenetic regulation, it is unclear how these cytosine modifications are reversed. Here, we demonstrate that 5mC and 5hmC in DNA are oxidized to 5-carboxylcytosine (5caC) by Tet dioxygenases in vitro and in cultured cells. 5caC is specifically recognized and excised by thymine-DNA glycosylase (TDG). Depletion of TDG in mouse embyronic stem cells leads to accumulation of 5caC to a readily detectable level. These data suggest that oxidation of 5mC by Tet proteins followed by TDG-mediated base excision of 5caC constitutes a pathway for active DNA demethylation.

Fig. 1. Purified Tet2 catalyzes the modification of 5mC and 5hmC

(A) The ³²P spot X on a TLC plate generated from 5mC and 5hmC DNA substrates incubated with the full-length Flag-Tet2 protein. The top spot in lanes 3 to 8 was 5′ end-labeled deoxyadenosine monophosphate (dAMP) resulting from the incomplete EcoNI digestion of the DNA substrate (fig. S1A). (B) TLC confirmation of the origin of spot X from 5mC with a ¹⁴C-labeled methyl group. (C) HPLC detection of a new nucleoside generated from 5mC and 5hmC DNA substrates upon incubation with Flag-Tet2. AU indicates absorption units.

Fig. 2. The modification product of 5mC is 5caC

The 5mC derivative generated by Flag-Tet2 was analyzed by HPLC analysis (A), TLC (B), and mass spectrometry (C) using a 5caC standard as a reference. (A) HPLC analysis of the nucleoside derived from 5mC in the DNA substrate treated with Flag-Tet2. (B) TLC identification of the modification product of Tet2. The 5mC substrate used was the same as in Fig. 1A. 5caC DNA (lane 3), a synthetic oligonucleotide duplex with the same sequence but containing a 5-caC 3′ of the cleavage site of EcoNI, provides a reference spot for 5-carboxycytidine monophosphate (5ca-dCMP). (C) Mass spectrometry analysis of a HPLC fraction corresponding to the peak X′ in (A). Structural formulas deduced are shown for the major peaks.

Fig. 3. Tet2 catalyzes formation of 5caC in genomic DNA in vivo

(A) HPLC detection of 5caC in genomic DNA of HEK 293T cells expressing wild-type Flag-Tet2. Synthetic 2′-deoxy-5-carboxylcytidine (5caC) and the nucleoside hydrolysate of a synthetic DNA containing cytosine (C), 5hmC, and 5mC were used as standards. Arrows point to the 5caC peaks detected in DNA isolated from cells transfected with wild-type Tet2. (B) HPLC–tandem mass spectrometry (MS/MS) detection of genomic 5caC. Shown are MRM elution profiles of negative-ion mass transitions from a precursor to its three product ions as shown in Fig. 2C for a synthetic 2′-deoxy-5-carboxycytidine standard (blue) and a DNA hydrolysate [isolated peak indicated with a red arrow in (A)] from cells transfected with full-length Tet2 (black). Red line was from a control DNA sample isolated from cells transfected with the inactive Tet2 mutant.

Fig. 4. TDG glycosylase recognizes and excises 5caC from DNA

(A) ES cell nuclear extract contains 5caC-specific base-excision activity. The activity in the nuclear extract of mouse ES cells to generate alkaline-sensitive sites was assayed by using 5caC-containing oligonucleotide duplexes. Shown are the results obtained with 20-mer DNA duplexes containing either G/U (U), G/5hmC (5hmC), or G/5caC (5caC) base pairs in the middle. (B) Excision of 5caC from DNA by Flag-TDG but not by Flag-MBD4, Flag-UNG, or GST-SMUG1. Asn¹⁵¹→Ala¹⁵¹ (N151A) is a catalytically inactive mutant of TDG. Proteins used are shown in fig. S10B. 6xHis-TDG purified from bacteria was also active in 5caC excision. TDG displayed a much stronger glycosylase activity for the “hemi-carboxylated” DNA substrate containing 5caC only on one strand (fig. S11). WT, wild type. (C) Reduced 5caC formation by cotransfection of TDG in HEK 293T cells expressing ectopic Tet2. The mutant TDG was as in (B). (D) Lack of 5caC base-excision activity in Tdg knockdown ES cells. Nuclear extracts prepared from the two independent cell lines (a and b) containing shRNA knockdown construct 1, 2, or scramble control were tested as in (A). (E) HPLC-MS/MS detection of 5caC in ES cells depleted of TDG. MRM profiles of hydrolysates of genomic DNA from control (red) and TDG-depleted ES cells (pink) were analyzed. Synthetic 5caC nucleoside was used as a positive control (blue). Depletion of TDG was confirmed by Western analysis of independent stable knockdown ES cell lines (fig. S12).