Figure 6: Loss of Tet2 and Tet3 results in DNA damage and impaired DNA repair.

(a–c) γH2AX levels in whole-cell lysates from bone marrow (top) and spleen (bottom), assessed by immunoblotting. Actin serves as a loading control. (a) Progressive increase in γH2AX levels in bone marrow and spleen of Tet2/3 DKO mice with time after pIpC injection. (b) Accumulation of γH2AX in bone marrow and spleen of Tet2/3 DKO but not Tet2KO or Tet3KO mice, 3 weeks after pIpC injection. (c) Impaired DNA repair in Tet2/3 DKO cells. WT and DKO mice were exposed to 6 Gy of ionizing radiation 3 weeks after pIpC injection, single-cell suspensions of bone marrow and spleens were prepared at the indicated times, and DNA repair kinetics were assessed by immunoblotting. Similar results were obtained 1 week after pIpC injection (Supplementary Fig. 19a). (d–g) DNA damage repair is impaired in Tet2/3 DKO myeloid-lineage cells. LSK (d), GMP (e) and Mac-1⁺ cells in bone marrow (f), or Mac-1⁺ cells in spleen (g) were sorted from WT and DKO mice at 3 weeks after pIpC injection, exposed to 6 Gy of ionizing radiation, and DNA repair kinetics were assessed by immunocytochemistry. LSK cells repair DNA damage efficiently, whereas GMP and Mac-1⁺ cells do not. Similar results were obtained using tamoxifen-injected Tet2^fl/fl Tet3^fl/fl ERT2-Cre⁺ mice (Supplementary Fig. 19b–d). Scale bar, 5 μm. (h) Increase in γH2AX levels in Mac-1⁺ cells sorted from bone marrow of DKO mice 3–4 weeks after pIpC injection. (i) TET proteins control expression of DNA repair genes in myeloid cells. Quantitative reverse transcription–PCR of Mac-1⁺ cells sorted from bone marrow of WT (Tet2^fl/fl Tet3^fl/fl) and DKO (Tet2^fl/fl Tet3^fl/fl Mx1-Cre⁺) mice 3–4 weeks after pIpC injection. Results are expressed as fold change over WT cells (set to 1; three independent experiments; means±s.e.m.). Similar results were observed in sorted Mac-1⁺ cells from WT (Tet2^+/+Tet3^fl/fl) and Tet2^−/− Tet3^fl/fl Mx1-Cre⁺ mice (Supplementary Fig. 19e). HR, homologous recombination; NHEJ, non-homologous end-joining; ND, not detected. *P<0.05, **P<0.005, ***P<0.0005 (Student's t-test).