SETD1A-deficieny results in defective DNA repair-mediating increased radiosensitivity in vivo. (A-F) KSL cells 7 to 9 weeks after TAM treatment from SCL-CreER^T+;Setd1a^F/F and control animals were analyzed for DNA damage-signaling and repair ex vivo and after irradiation (10 Gy). (A) Representative FACS of KSL cells for phosphorylated histone 2A.X at Ser139 (γH2A.X). (B) Graphs showing the MFI for γH2A.X ex vivo (left) and directly after irradiation (second from left). The percentages of γH2A.X⁺ cells at 1 hour (second from right) and at 2 hours (right) after irradiation are shown (F/F, n = 5; +/+ n = 4). Data are from 2 independent experiments. (C) Representative FACS of KSL cells for DNA breaks detected by TUNEL assay before and after irradiation. (D) Graphs showing the MFI for TUNEL label ex vivo (left) and directly after irradiation (second from left). The percentages of TUNEL⁺ cells at 1 hour (second from right) and at 2 hours (right) after irradiation are shown (F/F, n = 4; +/+ n = 2). Data are from 2 pooled and 3 individual SCL-CreER^T+;Setd1a^F/F mice and from pooled SCL-CreER^T+;Setd1a^+/+ mice from 2 independent experiments (3 and 4 mice). (E) Comet assay to detect DNA breaks (20× objective; Vista Green DNA dye supplied in the Kit). Undamaged cells (category I, left) and significantly damaged cells (hedgehog shape, category II, left) were scored. (F) Frequencies of KSL cells in category I or II without irradiation (left), 1 hour after irradiation (middle), and 2 hours after irradiation (right) (F/F n = 4, ^+/+ n = 4). (G) Survival of TAM-treated (9 and 12 weeks beforehand) SCL-CreER^T+;Setd1a^F/F mice (red line, n = 12), SCL-CreER^T+;Setd1a^F/+ mice (light blue line, n = 7), and SCL-CreER^T+;Setd1a^+/+ mice (blue line, n = 9) after irradiation with 6.6 Gy. Data are pooled from 2 independent experiments. (H) Donor cell contribution to blood PMNs after transplantation of 3 × 10⁶ wild-type Lin⁻ BM cells into nonconditioned SCL-CreER^T+;Setd1a^F/F (red line, n = 5) or SCL-CreER^T+;Setd1a^F/+ (blue line, n = 5) recipient mice that were TAM induced 7 weeks before. Data are representative of 2 independent experiments. (I) Wild-type donor-cell contribution to the LT-HSC compartment of nonconditioned SCL-CreER^T+;Setd1a^F/F or SCL-CreER^T+;Setd1a^F/+ recipients 16 weeks after transplantation.