Extended Data Fig. 2: Deamidation of H1 N76/77 is increased after DNA damage.
From: Histone H1 deamidation facilitates chromatin relaxation for DNA repair
a, Dot blot analysis of the indicated amounts of peptides using a purified H1N76/77D antibody diluted 1:1,000. The sensitivity of the purified anti-H1N76/77D antibody was <1 ng. Dot blot using indicated peptides showing that anti-H1N76/77D only recognized 2ND peptides, not WT peptides. b, ELISA confirmation that the specificity of the purified anti-H1N76/77D antibody against the immunogens was 27-fold higher than of unmodified (WT) peptide. c, WCLs of H1.4-KO HeLa cells with stable expression of WT or mutant FLAG-H1.4 were extracted, immunoprecipitated using anti-FLAG, and analyzed by immunoblotting. The H1N76/77D antibody specifically recognized double-deamidated H1 and did not interact with single-deamidated H1.4. d, H1.4-KO HeLa cells with stable expression of H1.4-WT or vector control were treated with 10 Gy IR. WCLs were extracted, immunoprecipitated using anti-FLAG, and analyzed by immunoblotting. The H1N76/77D antibody specifically recognized double-deamidated H1 and did not interact with FLAG-Vector immunoprecipitants. e, WT or quadruple H1 variant-knockout (H1.1-H1.4 KO; 4KO) HeLa cells were micro-irradiated and analyzed by immunofluorescence at 5 min post-irradiation. Scale bars, 5 μm. f, WT or 4KO HeLa cells were exposed to 10 Gy IR and released for 5 min. Histones were extracted for immunoblotting. g, HeLa cells were exposed to the indicated doses of IR and released for 2 min (top), or exposed to 10 Gy IR and released for the indicated times (bottom). Histones were extracted for immunoblotting. h, HeLa cells were exposed to the indicated doses of VP16 for 2 h (top) or exposed to 20 μM VP16 for 2 h and released for indicated times (bottom). Histones were extracted for immunoblotting. i, U2OS-AsiSI-ER-AID cells were treated with or without 4OHT (500 nM) for 4 h and subjected to chromatin immunoprecipitation (ChIP) with anti-IgG or anti-H1N76/77D followed by real-time PCR analysis. j, HeLa cells were exposed to ultraviolet radiation C (UVC, 20 J/cm2), then released for the indicated times. Histones were extracted for immunoblotting. k, HeLa cells were exposed to 8 mM hydroxyurea (Hu) for 3 h, 0.2 mM palmitic acid (PA) for 24 h, or 0.1 μg/mL TNF-α for 5 h. Histones and WCLs were extracted for immunoblotting. Data represent the means ± s.d. n = 3 samples (i) from three independent experiments. P values were calculated using Student unpaired two-tailed t-tests (i). Three independent experiments were performed (a-k).
