Fig. 4: H1(N76D/N77D) promotes p300 binding to chromatin and H1K75ac.
From: Histone H1 deamidation facilitates chromatin relaxation for DNA repair
a, HeLa cells were transfected with the indicated siRNAs, followed by plasmid transfection for 48 h, and exposed or not to 10 Gy IR. Histones and WCLs were analysed by immunoblotting. b, HeLa cells were transfected with Flag–H1.4 for 48 h and exposed or not to 10 Gy IR. WCLs were extracted, immunoprecipitated using anti-Flag and analysed by immunoblotting. c, Recombinant GST or GST–p300 fragments (top) were incubated with His–H1.4 for GST pull-down assays (bottom). Coomassie brilliant blue (CBB) staining is shown. The asterisks indicate specific protein bands. FL, full-length. d, GST or GST–p300 HAT was subjected to in vitro acetylation assays using free histones extracted from HeLa cells. e–g, H1.4-KO HeLa cells stably expressing wild-type or mutant Flag–H1.4 were modified to express FKBP–CTPS1, treated with dTAGV-1 for 3 h, and exposed or not to 10 Gy IR. Chromatin was analysed by immunoblotting (e), or proximity ligation assay was performed using anti-H1.4 and anti-p300, with representative images (f) and quantification of 50 cells (g) shown. Scale bars, 5 μm. h, Recombinant GST–p300 HAT protein was incubated with His–H1.4, His–H1.4-2ND, His–H1.4-2NA or His–H1.4-2NR for the GST pull-down assay (top). CBB staining is also shown (bottom). The asterisks indicate specific protein bands. Exp., exposure. Data represent the mean ± s.d. P values were calculated using Student’s unpaired two-tailed t-tests (g). n = 50 cells from 3 independent experiments (g).
