Genomic DNA of TET1-overexpressing cells contains a modified nucleotide within the dinucleotide CG. (A) Description of experimental design. (B to F) Genomic DNA was purified from HEK293 cells overexpressing TET1 and cleaved with [(B) and (C)] MspI, (D) HpaII, or [(E) and (F)] Taq^αI. The fragments were end-labeled, digested to 5′ dNMPs, and resolved by TLC. A modified nucleotide (subsequently identified as hm-dCMP) is indicated by ?. Neither 5m-dCMP nor the modified nucleotide are observed when the DNA is digested with HpaII. [(C) and (F)] Quantification of the relative abundance of dCMP, 5m-dCMP, and the modified nucleotide. Data shown are mean ± SD of three independent transfections and are representative of at least three experiments.

Recombinant Flag-HA-TET1-CD purified from Sf9 cells converts 5mC to hmC in methylated DNA oligonucleotides in vitro. (A) Double-stranded DNA oligonucleotides containing a fully methylated Taq^αI site were incubated with wt or mut Flag-HA-TET1-CD (1:10 enzyme to substrate ratio). Recovered oligonucleotides were digested with Taq^αI and analyzed by TLC. The faint dCMP spot in each lane is derived from end-labeling of the C at the 5′ end of each strand of the substrate. (B) The extent of conversion of 5mC to hmC is shown as the mean ratio [hmC/(hmC+5mC)] ± SD. (C) Comparison of species at an Rf of 0.29 in products resulting from incubation with wt or mut Flag-HA-TET1-CD. (D) MS fragmentation analysis of authentic hm-dCMP (top) and the nucleotide generated by Flag-HA-TET1-CD (bottom). Observed masses are shown in black (mass accuracy was within 0.002 Da). (E) Recombinant Flag-HA-TET1-CD is able to hydroxylate 5mC in fully methylated (full, lanes 5 and 6) and hemimethylated (hemi, lanes 3 and 4) substrates. Unme, unmethylated DNA oligonucleotide (lanes 1 and 2).

Expression of TET1 in HEK293 cells results in decreased 5mC staining. (A) Predicted domain architecture of human TET1 showing the CXXC-type zinc-binding domain (amino acid 584 to 624); the cysteine-rich region (Cys-rich) (amino acid 1418 to 1610); the DSBH domain (amino acid 1611 to 2074); and three bipartite nuclear localization sequences (NLS). (B) HEK293 cells overexpressing wild-type or mutant HA-TET1 were costained with antibodies specific for the HA epitope (green) and 5mC (red). To orient the reader, some HA-expressing cells are circled. Scale bar, 10 μm. (C) Staining intensities of HA and 5mC were measured in individual nuclei. Data from HA-TET1–expressing cell populations are presented as dot plots (red), superimposed on dot plots from mock-transfected cells (blue), with each dot representing an individual cell. (D) Quantification of 5mC staining intensity of HA-positive cells compared with that of mock-transfected cells (set to 1). Data shown are mean ± SEM and are representative of three experiments.

The modified nucleotide is identified as 5-hydroxymethylcytosine. (A) Genomic DNA from T4 phage grown in UDP-glucose–deficient E. coli ER1656 (T4*) and HEK293 cells transfected with wild-type or mutant TET1-CD were digested with Taq^αI. The fragments were end-labeled, digested to mononucleotides, and separated by TLC. The modified nucleotide present in TET1-CD–expressing cells migrates similarly to authentic hm-dCMP. (B) Comparison of liquid chromatography–electrospray ionization MS ions present at an Rf = 0.29 in genomic DNA from cells expressing wild-type (wt) or mutant (mut) TET1-CD. A species with an observed m/z = 336.06 was more abundant by a factor of 18.5 in the wild-type sample compared with the mutant sample. (C) Mass spectrometry fragmentation (MS/MS) analysis of authentic hm-dCMP (top), and the m/z = 336.06 species isolated from cells expressing TET1-CD (bottom). Expected m/z values are shown in red; observed m/z values are shown in black (anticipated mass accuracy is within 0.002 Da).

hmC is present in ES cell DNA, and its abundance decreases upon differentiation or Tet1 depletion. (A) (Left) TLC showing that hmC is detected in the genome of undifferentiated ES cells but not dendritic cells or T cells. (Right) Quantification of the relative abundance by PhosphorImager of 5mC, C, and hmC in the genomic DNA of undifferentiated ES cells. (B) Tet1 mRNA levels decline by ∼80% in ES cells induced to differentiate by withdrawal of LIF for 5 days. (C) The same differentiated ES cells show ∼40% decrease in hmC levels. (D) Transfection of ES cells using two different RNAi duplexes directed against Tet1 decreases Tet1 mRNA levels by ∼75%. (E) The same Tet1-depleted ES cells show ∼40% decline in hmC levels. Data shown are mean ± SD and are representative of 2 to 3 experiments.