Extended Data Fig. 3: CTPS1 deamidates H1 N76/77 in response to DNA damage.
From: Histone H1 deamidation facilitates chromatin relaxation for DNA repair
a, HeLa cells were transfected with control siRNA (siCtrl) or siRNA targeting individual GATs for 48 h and then exposed or not to 10 Gy IR. Histones were extracted for immunoblotting (top). RNA from cells without IR was extracted for real-time PCR to validate the knockdown efficiency (bottom). b, HeLa cells expressing FKBP-CTPS1 were treated with dTAGV-1 (1 mM) for 3 h and then exposed to 10 Gy IR. WCLs or histones were extracted for immunoblotting after 2DGE (left) or regular SDS-PAGE (right). c,d, HeLa cells were transfected with FLAG-CTPS1 (c left) or FLAG-H1.4 (c right) with or without 10 Gy IR (c) or all exposed to 10 Gy IR (d). WCLs were immunoprecipitated using anti-FLAG (c) or anti-IgG, anti-H1.4, or anti-CTPS1 (d) and analyzed by immunoblotting. e, HeLa cells were transfected with FLAG-CTPS1 for 48 h and exposed or not to 10 Gy IR. WCLs were extracted, immunoprecipitated with anti-FLAG, and analyzed by immunoblotting. The relative quantifications of the binding intensities are indicated below each band. f, GST or GST-H1.4 recombinant proteins were incubated with HIS-CTPS1 for GST pull-down assays; *indicates specific protein bands. g, Histone octamer and GST-H1.4 recombinant proteins were mixed and incubated with HIS-CTPS1 for pull-down assay using HIS beads (top). CBB staining shows the input (bottom). h, HIS-H1.4-WT purified from E. coli was incubated with or without FLAG-CTPS1-WT or FLAG-CTPS1-ED purified from HEK293T cells. Immunoblots of in vitro deamidation reactions analyzed after 2DGE (left) and regular SDS-PAGE (right) are shown. i, HeLa cells were exposed to the indicated doses of IR and released for 2 min (left), or exposed to 10 Gy IR and released for indicated times (right). Chromatin was extracted for immunoblotting. j, HeLa cells were exposed to 20 μM VP16 for 2 h and released for the indicated times (top) or to the indicated doses of VP16 for 2 h (bottom). Chromatin was extracted for immunoblotting. k, HeLa cells were micro-irradiated, fixed at 5 min post-irradiation, and analyzed by immunofluorescence using anti-CTPS1 (red) and anti-γH2AX (green). l,m, U2OS-265 reporter cells were transfected with GFP-CTPS1 plasmid (left) or GFP vector (right) for 48 h and treated with 4OHT (500 nM) and shield Ι (30 ng/mL) for 6 h. Co-localization of FokI-mCherry (red) and GFP-CTPS1 (green) was analyzed by immunofluorescence (l). Quantifications of 40 cells from three independent experiments are shown (m). n, WT or 4KO HeLa cells were exposed to 10 Gy IR and released for 5 min. Chromatin fractions were extracted for immunoblotting. o,p, WT or 4KO HeLa cells were transfected with GFP-CTPS1 for 48 h, then micro-irradiated. Images were captured every 10 s for 5 min. Representative images (o) and quantification of 30 cells from three independent experiments (p) are shown. Data represent the means ± s.d. (a, p) and values (m). n = 3 samples (a), n = 40 cells (m) or n = 30 cells (p) from three independent experiments. P values were calculated using Student unpaired two-tailed t-tests (a). Scale bars, 5 μm (k,l,o). Three independent experiments were performed in (a-k,m,n).
