Fig. 3: H1(N76D/N77D) is a prerequisite for H1K75ac.
From: Histone H1 deamidation facilitates chromatin relaxation for DNA repair
a, Chromatin fractions were extracted from wild-type or H1.4-KO HeLa cells stably expressing wild-type or mutant Flag–H1.4 and analysed by an MNase-sensitivity assay (top). WCLs were analysed by immunoblotting (bottom). b, 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 using anti-H1K75ac or anti-H1K75ac2ND antibodies. c, H1.4-KO HeLa cells stably expressing wild-type or mutant Flag–H1.4 were exposed to IR. WCLs were extracted, immunoprecipitated using anti-Flag and analysed by immunoblotting. d,e, Wild-type or CTPS1-KO HeLa cells were analysed by immunofluorescence for H1 PTM (red) and γH2AX (green) at 5 min post-micro-irradiation. Representative images (d) and quantification (e) of 30 cells are shown. Scale bars, 5 μm. f,g, Wild-type or H1.4-KO HeLa cells stably expressing wild-type or mutant Flag–H1.4 were analysed by a comet assay at the indicated times post-IR (f) or post-VP16 (g). Statistical analyses of the tail moment at the indicated time points post-treatment are shown. h,i, Wild-type or H1.4-knockout HeLa cells stably expressing wild-type or mutant Flag–H1.4 were analysed by immunofluorescence for RAD51 (h) or 53BP1 (i) foci at the indicated times post-IR. Statistical analyses of foci numbers at the indicated time points post-IR are shown. Data represent the mean ± s.d. P values were calculated using Student’s unpaired two-tailed t-tests (e–i). n = 30 (e) and n = 50 (f–i) cells from 3 independent experiments.
