Cell culture
HeLa, HCT116, A549, H1975, HepG2, Hepa1-6, NCI-H1299, HCC1937, MCF7, T47D-KBluc, 786-O, A-498, 5637, T24 and HEK293T cells were obtained from the American Type Culture Collection. AsiSI-ER-U2OS-AID cells were provided by G. Legube; PARP1-KO, EJ5-U2OS and DR-U2OS cells were provided by X. Xu; and U2OS-265 reporter cells were provided by R. Greenberg. All cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin, and maintained in a humidified incubator at 37 °C under 5% CO2 and were free of mycoplasma contamination.
Plasmid construction and transfection
H1.4 (also known as H1-4) and CTPS1 cDNAs were cloned from HeLa cells and subcloned separately into p3×Flag-CMV10, EGFP-C1, pET28b and pGEX-4T3 vectors. The cDNAs for p300 (also known as EP300), CBP (CREBBP), hMOF (KAT8), TIP60 (KAT5) and PCAF (KAT2B) were cloned into p3×Flag-CMV-10. The cDNAs for p300 HAT were cloned into p3×Flag-CMV-10, mCherry-N1, EGFP-C1 and pGEX-6P1 vectors. The cDNA for p300 fragments were cloned into pGEX-6P1. The cDNAs for H1.1, H1.2 H1.3 and H1.5 were cloned into p3×Flag-CMV-10. All mutant constructs were generated using the wild-type construct as a template with a Mut Express II Fast Mutagenesis Kit (Vazyme Biotech) according to the manufacturer’s protocols. A list of the primers for plasmid construction is provided in Supplementary Table 1.
For transient transfections, cells were transfected with the above mentioned plasmids using UltraFection 3.0 reagent (4A Biotech), following the manufacturer’s instructions. Cells were also transfected with either non-targeting control or gene-specific siRNAs using INTERFERin transfection reagent (Invitrogen), according to the manufacturer’s guidelines. All siRNA sequences are listed in Supplementary Table 2. Homozygous insertions and knock-ins were confirmed by Sanger sequencing and western blotting. A list of the oligos and sequences for the single guide RNAs (sgRNAs) is provided in Supplementary Table 3.
Stable cell line establishment
For knockout cell lines, pLentivirus2 plasmids carrying CRISPR–Cas9 and an sgRNA targeting H1.1–H1.4 or CTPS1 were transfected into HEK293T cells for 72 h using polyethylenimine (Polysciences) according to the manufacturer’s instructions. The culture medium containing the lentivirus was collected and transferred to HeLa cells in the presence of 8 μg ml−1 polybrene. After infection for 48 h, cells were sub-seeded into 96-well plates and selected for single colonies using puromycin (2 μg ml−1) for 1 week. Proteins were extracted from WCLs of surviving colonies for western blot analysis.
For overexpressing cell lines, the pCDH-CMV-MCS-EF1 lentivirus carrying sgRNA-resistant mutated Flag–H1.4 was transfected into H1.4-KO cells for 48 h. The lentivirus carrying sgRNA-resistant Flag–CTPS1-WT or ED was transfected into CTPS1-KO cells for 48 h. The transfected cells were then cultured in medium containing puromycin (5 μg ml−1) for 10 days. After antibiotic selection, cells were sub-seeded into 96-well plates for selecting single colonies. Proteins were extracted from WCLs of surviving colonies for western blot analysis.
Knock-in cell line establishment
An FKBP12 degron tag-mediated system to deplete CTPS1 (FKBP–CTPS1) in HeLa cells was established as previously described61. To generate the endogenous dTAG-inducible degradation system for CTPS1, sgRNA targeting the start codon region and pUC19 plasmid were co-transfected into the HeLa cells or H1.4-KO HeLa cells expressing wild-type or mutant Flag–H1.4. The pUC19 plasmid contained the following elements: left and right homology arms, as well as FKBP12(F36V), 3×Flag tags, P2A and a gene encoding blasticidin-S deaminase (BSD). The transfected cells were selected with 10 µg ml−1 blasticidin-S-selective antibiotic (blasticidin-S HCl; A1113903, Thermo Fisher Scientific), and sub-seeded into 96-well plates for selecting single colonies and screened for correct biallelic integration. All homozygous insertions and knock-ins were confirmed by PCR and western blotting.
Subcellular fractionation
For histone acid extraction, cell pellets were lysed on ice in hypotonic buffer containing 0.4 M sulfuric acid and precipitated with trichloroacetic acid at 4 °C overnight. The histone pellet was washed twice with ice-cold acetone and then air-dried. The pellet was then resuspended in an equal volume of ddH2O and 2× loading buffer and boiled for 5 min before immunoblotting.
For chromatin fractionation, cells were harvested and lysed on ice in buffer I (50 mM HEPES pH 7.5, 150 mM NaCl and 1 mM EDTA) containing 0.1% Triton X-100 and 1% protease inhibitor cocktail for 3 min. After centrifugation (13,000g for 3 min), the supernatant was discarded, and the pellet was resuspended on ice in buffer I containing 200 μg ml−1 RNaseA and 1% protease inhibitor cocktail for 3 min. After a second centrifugation, the pellet was resuspended in an equal volume of PBS and 2× loading buffer and boiled for 5 min before immunoblotting.
For WCL extraction, cells were harvested and lysed on ice for 30 min in lysis buffer (20 mM Tris-HCl pH 7.5, 137 mM NaCl, 10% glycerol, 1% NP-40, 2 mM EDTA and 1% protease inhibitor cocktail), followed by centrifugation at 13,000g at 4 °C for 15 min. After centrifugation, the pellet was resuspended in 5× loading buffer and boiled for 5 min before immunoblotting.
Co-immunoprecipitation
For endogenous co-immunoprecipitation, WCLs were prepared on ice for 30 min in lysis buffer (20 mM Tris-HCl pH 7.5, 137 mM NaCl, 10% glycerol, 1% NP-40, 2 mM EDTA and 1% protease inhibitor cocktail), followed by centrifugation at 13,000g at 4 °C for 15 min. The lysates were immunoprecipitated with the indicated antibodies (2 μg) overnight at 4 °C, followed by the addition of 30 μl protein G or protein A agarose gel beads. After incubation for a further 2 h at 4 °C, the beads were washed three times in NP-40 buffer (20 mM Tris-HCl pH 8.0, 137 mM NaCl, 1% NP-40, 10% glycerol, 2 mM EDTA and 1% protease inhibitor cocktail) and centrifuged at 100g at 4 °C for 1 min. The precipitated components were boiled and analysed by western blotting.
For Flag co-immunoprecipitation, WCLs were extracted and immunoprecipitated with Flag–agarose gel beads overnight at 4 °C. The beads were washed three times in NP-40 buffer and centrifuged at 100g at 4 °C for 1 min. The immunoprecipitated proteins were eluted overnight at 4 °C with 50 μl 3× Flag peptide (Sigma-Aldrich; 0.125 mg ml−1) in TBS buffer (50 mM Tris-HCl, pH 7.4, and 150 mM NaCl). The supernatant was boiled with 2× SDS loading buffer (100 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 0.2% bromophenol blue and 2% β-mercaptoethanol) for western blotting.
Protein purification and GST pull-down
His-tagged or GST-tagged plasmids were transformed into E. coli BL21 cells and induced with 0.1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) overnight at 16 °C, followed by purification using an Ni-IDA Sepharose gel or glutathione–Sepharose 4B beads, respectively. Equal amounts of individual His-fusion proteins were incubated for 4 h at 4 °C with GST-fusion proteins in TEN buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA and 100 mM NaCl). The beads were then washed three times in TEN buffer with centrifugation at 100g at 4 °C for 1 min and boiled for 5 min in an equal volume of 2× SDS loading buffer before immunoblotting or Coomassie brilliant blue staining.
Western blot analysis
Equivalent amounts of boiled protein samples were separated by SDS–PAGE (6–15% gels) and transferred to nitrocellulose membranes. After blocking with 5% BSA, the membranes were incubated overnight at 4 °C with the indicated primary antibodies and then with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. The proteins were detected using an ECL kit (WBULS0500, Millipore-Sigma Aldrich) and visualized with a Tanon 5200SF Imaging System. A list of information on antibodies is provided in Supplementary Table 4.
Mass spectrometry
For H1 PTM identification, histones were separated by SDS–PAGE and stained with Coomassie brilliant blue. The bands were then sent to Wininnovate Bio for liquid chromatography–tandem mass spectrometry to identify H1 PTMs. The desired PAGE bands were destained, dehydrated and digested in the gel in mass spectrometry-grade trypsin (V5280, Promega Biotech) for 12 h at 37 °C. Peptides were extracted from the gels with 70% acetonitrile/water containing 0.5% trifluoroacetic acid, then lyophilized and resuspended in 5% acetonitrile/water (0.1% formic acid), before 6 μl of the peptide mixture was loaded onto a nanoViper C18 (Acclaim PepMap 100, 75 μm × 2 cm) trap column. The peptides were chromatographically separated on an Easy nLC 1200 system (Thermo Fisher). Tandem mass spectra were acquired on a Q Exactive mass spectrometer (Thermo Fisher) equipped with a Nano Flex ion source. The tandem mass spectrometry raw data were processed for protein identification and PTM analysis using PEAKS Studio 8.5 (Bioinformatics Solutions). The database search parameters were set as follows: proteome database of Uniprot-human including 20,603 protein entries or target protein of H1.2_HUMAN Histone (Uniprot accession number: P16403); mass tolerance for precursor and fragment ions at 10 ppm and 0.05 Da, respectively; variable modifications of acetylation (protein-N terminus, K), oxidation (M), deamidation (NQ) and fixed modification of carbamidomethylation (C). Label-free quantification analysis of PTMs between different samples was performed based on the peptide area in each sample, calculated by distributing the associated peptide feature area, including different m/z-extracted ion chromatograph areas.
For CTPS1 phosphorylation site identification, WCLs were extracted, immunoprecipitated using anti-Flag, and separated by SDS–PAGE and stained with Coomassie brilliant blue. The bands were then sent to PTM Bio for liquid chromatography–tandem mass spectrometry to identify CTPS1 phosphorylation sites.
Two-dimensional gel electrophoresis
The extracted histone proteins were dissolved in 150 μl rehydration buffer (8 M urea, 2% CHAPS, 0.5% IPG buffer and 0.002% bromophenol blue), and then loaded onto isoelectric focusing (IEF) strips using the following programme: 20 V for 10 h (rehydration); 500 V for 1 h; 1,000 V for 1 h; 1,000–5,000 V for 4 h; and 5,000 V for 4 h. Following IEF, the strips were incubated for 15 min in SDS equilibration solution containing 10 mg ml−1 dithiothreitol (DTT) and then for another 15 min in SDS equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS and 0.001% bromophenol blue) containing 2-iodoacetamide. Strips were washed in SDS–PAGE buffer and resolved by SDS–PAGE before immunoblotting.
CUT&Tag-seq and ATAC-seq
U2OS-AsiSI-ER-AID cells were exposed to 500 nM 4OHT for 4 h. CUT&Tag or ATAC-seq was performed using the Hyperactive Universal CUT&Tag Assay Kit or Hyperactive ATAC-Seq Kit (Vazyme).
CUT&Tag was performed with anti-H1(N76D/N77D), anti-H1K75ac, anti-H1K75ac2ND or anti-CTPS1 antibodies. In brief, nuclei from 1 × 105 cells were extracted with the provided nuclei extraction buffer and then incubated with 10 μl ConA beads. Then, 1 μg anti-H1(N76D/N77D), anti-H1K75ac, anti-H1K75ac2ND or anti-CTPS1 antibodies was added and cultured for 2 h at room temperature. After washing twice with the provided dig-wash buffer, 0.5 μg secondary antibody was added and incubated at room temperature for 30 min. After washing twice with dig-wash buffer, 2 μl pA/G-Tn5 was cultured with the beads for 1 h, followed by tagmentation at 37 °C for 1 h. The reaction was stopped with proteinase K and buffer B/L, and DNA was extracted with DNA extraction beads for subsequent PCR to amplify the libraries.
For ATAC-seq, 5 × 104 cells were harvested and resuspended in the lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2 and 0.05% NP-40). The lysates were centrifuged for 3 min at 500g at 4 °C. The cell pellet was resuspended in transposition reaction mix and incubated at 37 °C for 45 min. The transposed DNA was purified using ATAC DNA extraction beads before PCR to amplify the libraries. The amplified ATAC-seq library was purified with ATAC DNA Clean Beads and eluted with 30 μl ddH2O.
Libraries were created using the TruePrep Index Kit (Vazyme) and sent to Novogene for sequencing on an Illumina NovaSeq 6000 platform. The raw reads were processed using Fastp (v0.23.4) to eliminate adaptors and low-quality sequences. Bowtie2 (v2.5.4) was applied for alignment to the hg38 human genome reference, using the parameters ‘–very-sensitive–no-mixed–no-discordant’. Samtools was used to convert SAM files to BAM format, applying a filter criterion of a minimum mapping quality score of 10. Duplicate reads were removed using the Picard tool. The bamCoverage function was then used to transform BAM files into BW format, with normalization applied using the parameter ‘–normalizeUsing BPM’. The top 80 BLESS signal enzyme-cutting sites62 were subjected to coordinate conversion from hg19 to hg38 using the liftOver utility. The generation of metagene profiles and heatmaps, which display the signals of each histone modification and open chromatin at DSBs, was accomplished using the computeMatrix and plotHeatmap functions of deepTools (v3.5.5). DSB sites were classified as prone to homologous recombination or NHEJ according to previous research62.
Comet assay
Cells were exposed to 10 Gy IR or 20 μM VP16 for 2 h. After release for the indicated time, cells were resuspended at a density of 5 × 105 per millilitre in ice-cold PBS and combined with molten low-melting-point agarose at a 1:10 (vol/vol) ratio. The cell–agarose mixture was spread onto pre-warmed comet slides and kept at 4 °C until the agarose solidified. The slides were then immersed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCl and 1% Triton X-100) overnight at 4 °C. The slides were incubated in freshly prepared running buffer (1 mM Na2EDTA and 300 mM NaOH) for 30 min, followed by electrophoresis in running buffer at 1.0 V cm−1 for 30 min. Slides were washed twice in 75% ethanol for 5 min and dried at 37 °C. Cells were then stained with 5 μg ml−1 propidium iodide for 30 min in the dark before images were captured using an Olympus BX51 fluorescent microscope. Comet tail moments were measured using the OpenComet plugin in ImageJ; 50 nuclei or 5 fields were captured and counted.
Enzyme-linked immunosorbent assay
The specificities of antibodies targeting H1(N76D/N77D), H1K75ac and H1 with spontaneous modifications of three sites (H1K75ac2ND) were validated by ELISA. The purified antibodies were incubated in 96-well plates precoated with 50 ng of modified or negative control peptides. The wells were then washed three times with PBS, and the absorbance of each well was measured at 450 nm using a microtitre plate reader.
Microscale thermophoresis
A total of 10 μl of 2 μM His–H1.4 or His–H1.4-KQ proteins purified from E. coli was mixed with 10 μl PBST binding buffer (PBS containing 0.05% Tween). After thorough mixing, 10 μl of the mixture was removed and serially diluted twofold. This dilution procedure was repeated 16 times as ligands. A total of 10 μl of 2 nM DNA-Cy5 were mixed with each ligand and then A Monolith NT.115 (NanoTemper Technologies) standard capillary was inserted into each mixture. The MST device was started after placing the capillaries in locations 1 through 16 in the sample tray. The data were analysed by plotting peptide concentrations against liquid-induced fluorescence changes (change in raw fluorescence on the y axis). Curve fitting was performed by using MO. Affinity analysis and the given Kd values were calculated with 95% confidence levels.
Immunofluorescent staining
Wild-type and CTPS1-KO HeLa cells, or H1.4-KO HeLa cell lines stably expressing mutant H1.4 (5 × 104) were seeded into a 35-mm glass-bottomed dish and cultured in a humidified incubator at 37 °C under 5% CO2 for 48 h. Cells were treated with 10 Gy IR or 20 μM VP16 for 2 h, and then fixed at indicated time with 1 ml of 4% tissue fixation solution at 4 °C for at least 30 min, washed three times with PBS, and then permeabilized with pre-cooled methanol at −20 °C for 30 min. After blocking with 5% BSA for 1 h at room temperature, the cells were washed and incubated overnight at 4 °C with the indicated antibodies. After washing three times with PBS, the cells were incubated with secondary antibodies conjugated to Alexa Fluor 488 or 594 for 1 h at room temperature. After washing three times with PBST, DAPI was used to stain nuclear DNA. Immunofluorescent images were captured under a Nikon confocal microscope.
In vitro acetylation assay
GST–p300 HAT was purified and incubated with substrates in acetylation buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl, 4 mM MgCl2, 0.1 mM EDTA, 1 mM DTT and 10% glycerol) with or without acetyl-CoA (5 mM), for 1 h at 30 °C. The reactions were stopped by adding 5× protein sample buffer (250 mM Tris-HCl pH 6.8, 10% SDS, 50% glycerol, 0.5% bromophenol blue and 5% β-mercaptoethanol), and the samples were boiled for 5 min before separation by SDS–PAGE and immunoblotting.
In vitro deamidation assay
Flag–CTPS1 was purified from transfected HEK293T cells, and His–H1.4 or GST–H1.4 was purified from E. coli cells. Flag–CTPS1 (8 μM) was incubated at 21 °C for 3 min in reaction buffer containing 20 mM Tris-HCl (pH 7.9) and 10 mM MgCl2. Flag–CTPS1 was then combined with pre-warmed (37 °C) nucleotides at final concentrations of 2 mM UTP, 2 mM ATP and 0.2 mM GTP for 30 min. The substrate His–H1.4 (2 μM) was added and incubated at 37 °C for an additional 45 min to allow for polymerization. The samples were then subjected to 2DGE and immunoblotting.
Laser micro-irradiation-coupled live-cell imaging
HeLa or HCT116 cells (7 × 104) were seeded into a 35-mm glass-bottomed dish and transfected with mCherry–p300 HAT, GFP–CTPS1 or GFP–PARP1 for 48 h. The cells were locally irradiated with a 365-nm pulsed nitrogen UV laser (16 Hz pulse, 41% laser output) generated from a MicroPoint system (Andor). This system was coupled directly to the epifluorescence path of the Nikon A1 confocal imaging system, and timelapse images were captured every 10 s for the indicated time. The intensity of the irradiation path signal from indicated cells was calculated using ImageJ software.
Laser micro-irradiation-coupled immunofluorescence
Wild-type or CTPS1-KO HeLa cells (3 × 105) were seeded into a 35-mm glass-bottomed dish and locally irradiated with a 365-nm pulsed nitrogen UV laser (16 Hz pulse, 41% laser output) generated from a MicroPoint system (Andor). Cells were fixed at 5 min post-irradiation with 1 ml of 4% tissue fixation solution at 4 °C for at least 30 min, washed three times with PBS, and then permeabilized with pre-cooled methanol at −20 °C for 30 min. After blocking with 5% BSA for 1 h at room temperature, the cells were washed and co-incubated overnight at 4 °C with anti-H1(N76D/N77D), anti-H1K75ac, anti-H1K75ac2ND or anti-CTPS1 and anti-γH2AX antibodies. After washing three times with PBS, the cells were incubated with secondary antibodies conjugated to Alexa Fluor 488 or 594 for 1 h at room temperature in the dark. After washing three times with PBST, DAPI was used to stain the nuclear DNA. Immunofluorescent images were captured under a Nikon confocal microscope.
RT–qPCR assay
Total RNA was extracted from cells using TRIzol reagent. The suspension was centrifuged (13,000g for 5 min), and the upper layer was precipitated using isopropanol and centrifuged at 13,000g for 10 min at 4 °C. After washing with 75% ethanol, the RNA was treated with DNase I for 30 min at 37 °C to remove contaminating DNA, and then reverse transcribed into cDNA using a QuantScript RT Kit (Tiangen) according to the manufacturer’s instructions.
ChIP–qPCR
U2OS-AsiSI-ER-AID cells (1.5 × 107) on 150-mm cell culture dishes were crosslinked using 1% formaldehyde for 10 min and quenched with 125 mM glycine for 2 min at room temperature. The cells were collected, washed twice with cold PBS and resuspended in immunoprecipitation buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 5 mM EDTA, 0.5% NP-40 and 1% Triton X-100) for ultrasound fragmentation. After centrifugation at 12,000g for 10 min at 4 °C, the supernatant was incubated with 1 μg normal rabbit IgG or anti-H1(N76D/N77D) antibody immobilized C on protein A/G Sepharose beads. The beads were washed five times with immunoprecipitation buffer and mixed with 100 μl of 10% Chelex (Bio-Rad). The samples were boiled for 10 min and the supernatants were collected. The pellets were resuspended in 120 μl of ddH2O and centrifuged at 10,000g for 1 min at 4 °C. The supernatants were combined as the IP DNA pool. The immunoprecipitation DNA pool and 2% input DNA were used as templates for qPCR analysis using SYBR Green Supermix (Vazyme) on a qTOWER3G Touch Real-Time PCR Detection System (Analytik Jena). All samples were analysed in triplicate.
ChIP–re-ChIP
U2OS-AsiSI-ER-AID cells (5 × 107) on 150-mm cell culture dishes were crosslinked using 1% formaldehyde for 10 min and quenched with 125 mM glycine for 2 min at room temperature. The cells were collected, washed twice with cold PBS and resuspended in IP buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 5 mM EDTA, 0.5% NP-40 and 1% Triton X-100) for ultrasound fragmentation. After centrifugation at 12,000g for 10 min at 4 °C, the supernatant was collected for the ChIP–re-ChIP assay using the Re-ChIP-IT kit (53016, Active Motif); 1% of the input DNA was stored as a control. In brief, chromatin was incubated with 3 μg H1.4 antibody or normal rabbit IgG and 25 μl protein G beads overnight at 4 °C. The beads were washed twice with both ChIP buffer 1 and ChIP buffer 2. The first ChIP reaction was eluted with 100 μl Re-ChIP-IT Elution buffer for 30 min at room temperature. The first ChIP chromatin was then collected with desalting columns and subjected to the second ChIP (containing 25 μl LSV protein G beads, 3 μg anti-H1(N76D/N77D), anti-H1K75ac or anti-H1K75ac-2ND antibodies or normal rabbit IgG) overnight at 4 °C. The beads were washed twice with both ChIP buffer 1 and ChIP buffer 2, and eluted with elution buffer AM2 for 15 min at room temperature. The chromatin was then reversed crosslinked and treated with proteinase K. The ChIP–re-ChIP DNA and input DNA were used as templates for qPCR analysis using SYBR Green Supermix (Vazyme) on a qTOWER3G Touch Real-Time PCR Detection System (Analytik Jena). All samples were analysed in triplicate.
In situ DSB reporter assay
265-U2OS cells (7 × 104) were seeded into a 35-mm glass-bottomed dish. The cells were transfected with GFP vector, GFP–p300 HAT or GFP–CTPS1 for 48 h before exposure to 4OHT (500 nM) and shield I (30 ng ml−1) for 6 h. The cells were then incubated with 1 ml of 4% tissue fixation solution at 4 °C for at least 30 min and washed three times with PBS. DAPI was used to stain nuclear DNA. Immunofluorescent images were captured under a Nikon confocal microscope.
Homologous recombination and NHEJ reporter assays
pDR-GFP-U2OS (homologous recombination) or pEJ5-GFP-U2OS (NHEJ) cells (7 × 104) were seeded into a 60-mm cell culture dish. The cells were transfected with CTPS1, p300 or control siRNA for 12 h, followed by transfection with Flag-tagged wild-type H1.4 or 2ND/2NR/2NA/KQ/KR mutants for an additional 12 h. The cells were then infected with a retrovirus expressing I-SceI for a further 48 h. After trypsinization, the cells were collected for flow cytometric analysis.
In situ proximity ligation assay
H1.4-KO HeLa cells (7 × 104) were seeded into a 35-mm glass-bottomed dish. The cells were exposed, or not, to 10 Gy IR, then incubated with 1 ml of 4% tissue fixation solution at 4 °C for at least 30 min. The cells were washed three times with PBS, followed by permeabilization with pre-cooled methanol at −20 °C for 30 min. After washing and blocking with 5% BSA for 1 h at room temperature, the cells were double-stained for PLA with anti-p300 and anti-H1.4 or anti-CTPS1 and anti-Flag using the Duolink In Situ Red Starter Kit (Sigma). Images were captured under a Nikon confocal microscope, and PLA foci were automatically quantified using ImageJ software.
MNase assay
MNase assays were performed as previously described63. In brief, HeLa cells (7 × 106) were treated with VP16 (40 μM) for 1 h. After centrifugation (1,500g for 5 min), the cell pellets were resuspended in buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM DTT, 0.1% Triton X-100 and protease inhibitor cocktail). After incubation on ice for 8 min, the lysates were centrifuged at 13,000g for 3 min at 4 °C. The pellet was further lysed in buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT and protease inhibitor cocktail) for 30 min on ice, followed by centrifugation at 1,700g for 5 min. The pellet was resuspended in MNase buffer (200 mM Tris-HCl pH 8.0, 50 mM NaCl and 25 mM CaCl2) with 10 U MNase for 1 min at 25 °C, and the reaction was stopped by adding 0.5 M EDTA. After incubation on ice for 10 min, RNase A and proteinase K were added. The DNA was separated by 1.2% agarose gel electrophoresis and the intensity of each lane was analysed using ImageJ.
Nucleosome stability assay
HeLa cells (7 × 106) were treated with VP16 (40 μM) for 1 h and then collected and resuspended in 500 μl buffer A (20 mM HEPES pH 7.9, 0.5 mM DTT, 1 mM PMSF, 1.5 mM MgCl2, and 0.1% Triton) containing either 1 M or the indicated concentrations of NaCl. Samples were then ultracentrifuged at 100,000g (Ultracentrifuge, Beckman Coulter) for 20 min, and the supernatant containing the released histones was collected for further analysis.
Chromosome aberration assay
Chromosome metaphase spreading assays were performed to investigate chromosomal abnormalities. The cells were exposed to 3 Gy IR and collected after 2 h of recovery and 8 h of pretreatment with colchicine (0.4 μg ml−1). After exposure to a hypotonic solution containing 56 mM KCl, the cells were preserved in a 3:1 methanol:acetic acid (v/v) solution. The cells were then dropped onto alcohol-cleaned slides and stained with Giemsa. Images were obtained using a DragonFly confocal imaging system (Andor); more than 100 mitotic chromosomes were randomly selected and examined in each experiment.
Colony formation assay
After 1 h of IR or VP16 treatment, equivalent numbers of cells (control group n = 500; experimental group n = 5,000) were seeded into 60-mm cell culture dishes and cultured for 2 weeks at 37 °C under 5% CO2. The cells were then stained with 1× crystal violet, and colonies containing more than 50 cells were counted.
Molecular docking
The AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk/) was used to predict the potential structure of CTPS1 (UniProt P17812). Histone H1.4 or H1.4-2ND were predicted by AlphaFold 2 (https://colab.research.google.com/AlphaFold2.ipynb).
The structure of p300 HAT was obtained from a previous report64 (https://www.rcsb.org). GRAMM (https://gramm.compbio.ku.edu) and PyMol were used for virtual verification of the binding pocket and hotspot amino acids of H1.4 with CTPS1, and H1.4 or H1.4-2ND with the p300 HAT. The structures of the binding between H1.4 and linker DNA were obtained from a previous report65 (https://www.rcsb.org). The Lys75, Asn76 and Asn77 amino acids were mutated by PyMol based on the reported wild-type structure. The binding between H1.4 or H1.4-QDD to linker DNA were visualized by PyMol.
Xenograft tumour model
Wild-type or CTPS1-KO HeLa cells, or H1.4-mutant HeLa cell lines with stable expression of Flag–H1.4-RRR or Flag–H1.4-RAA, were trypsinized, counted and resuspended at a 2:1 (v/v) ratio in PBS:Matrigel. The cells (5 × 106 in 100 μl of the mixture) were then seeded subcutaneously into 4-week-old female BALB/c nude mice (WuXi AppTec). The mice were randomly divided into IR therapy and control groups (six mice per group). For the therapy group, the tumour site was exposed to 3 Gy IR every 3 days, and the tumour size was measured after each treatment. Tumour volumes were assessed on the indicated days using Vernier calipers and calculated using the formula: volume = 0.5 × l × width2. Mice were euthanized (via cervical dislocation) after three or four treatments, and the tumours were weighed and stored in liquid nitrogen or fixed in formalin. The samples were thawed from liquid nitrogen, and WCLs or histones were isolated as described above. The protein samples were analysed by western blotting using the indicated antibodies. The use of animals in this study was approved by the Institutional Animal Care and Use Committee (The mechanisms of chromatin remodeling during early stage of DNA damage response, 202400110) of Shenzhen University.
Immunohistochemistry
Sections (5 μm) from formalin-fixed, paraffin-embedded archival tissues were deparaffinized, rehydrated and rinsed in distilled water. Antigen retrieval was achieved by incubating the slides with 1 mM EDTA buffer (pH 8.0) for 10 min in a pressure cooker. Endogenous peroxidase activity was blocked by incubating the slides in 3% hydrogen peroxide in methanol for 30 min. The sections were then stained for 16 h at 4 °C with polyclonal antibodies to CTPS1 and Ki-67 (1:100 and 1:500, respectively), and chromogen development was performed using the universal HRP Multimer UltraView Kit on a Benchmark XL (Ventana Medical Systems). Formalin-fixed, paraffin-embedded tissue sections were stained using a TUNEL Apoptosis Detection Kit (Yeasen) according to the manufacturer’s instructions. CTPS1 expression levels in tissue microarrays were evaluated using ImageJ software by considering the staining intensity and region.
Clinical patient samples
A total of 33 paired human cervical tumour and para-tumour samples were obtained from clinical patients diagnosed and treated with radiotherapies at Shanghai First Maternity and Infant Hospital. The samples were thawed from liquid nitrogen. WCLs were isolated using RIPA lysis buffer, and histone proteins were extracted in H2SO4. The protein samples were then analysed by western blotting using the indicated antibodies. A tissue microarray chip consisting of 58 cervical cancer samples from patients before radiotherapy was provided by Fudan University Shanghai Cancer Center. CTPS1 expression levels were determined by immunohistochemistry. All studies using human specimens were approved by the Clinical Research Ethics Committee of Shenzhen University. The patients provided written informed consent for the use of their resected tissues for research purposes. Data used for survival analyses in gastric and lung cancers were obtained from the KM Plotter website (https://www.kmplot.com/analysis/).
Antibody development
The anti-H1(N76D/N77D), anti-H1K75ac and anti-H1K75ac2ND antibodies were developed by PTM Bio. In brief, antigenic polypeptides were designed and synthesized for animal immunization (CYDVEKDDSRIK for anti-H1(N76D/N77D); CAGYDVE-(acetyl)K-NNSRIK for anti-H1K75ac; AGYDVE-(acetyl)K-DDSRIKC for for anti-H1K75ac2ND, immunization peptide). An unmodified control polypeptide was designed and synthesized for purification and detection purposes (CYDVEKNNSRIK for anti-H1(N76D/N77D); CAGYDVEKNNSRIK for anti-H1K75ac; AGYDVE-(acetyl)K-NNSRIKC and AGYDVEKDDSRIKC for anti-H1K75ac2ND). The polypeptides were conjugated with KLH for rabbit immunization. The immunogen was diluted with physiological saline and then mixed 1:1 with the corresponding adjuvant. The antigen and adjuvant were completely mixed to form a stable emulsion and then injected into six healthy New Zealand rabbits. Each rabbit was immunized four times. On the forty-fifth day, 30 ml of whole blood was collected and centrifuged. After centrifugation, the supernatant was collected for serum screening by ELISA and Dot blot. Positive serum samples were purified. Protein A column was packed and equilibrated with 10 volumes of pre-cooled PBS buffer solution. The filtered sample was loaded onto the equilibrated protein A column. The column was washed with PBS buffer and eluted with 150 mM glycine buffer. The crude IgG obtained from protein A purification was loaded onto the equilibrated antigen polypeptide affinity chromatography column to specifically enrich the target antibody. The target antibody obtained in the previous step was loaded onto an unmodified affinity chromatography column to remove nonspecific antibody components. The effluent was directly collected.
Statistics and reproducibility
GraphPad Prism 9.0.0 was used for data analysis. Each group of data was subjected to Kolmogorov–Smirnov tests, Anderson–Darling tests, D’Agostino–Pearson omnibus tests or Shapiro–Wilk tests for normal distribution when applicable. Student’s unpaired two-tailed t-tests were used to determine the significance between two selected groups of normally distributed data, unless indicated in the figure legends. P < 0.05 was considered statistically significant. For damaged versus undamaged chromatin feature comparisons, significant differences were determined using two-sample Wilcoxon tests. The correlation between protein expression levels was analysed using Pearson r test. Overall survival was analysed by log-rank (Mantel–Cox) test. Data are presented as the means ± s.d. All experiments were performed for at least three independent biological experiments unless indicated in the figure legends. Sample sizes were selected based on previous experience to ensure sufficient statistical power. For animal studies, power analyses were performed using a web-based tool (www.biomath.info). In vivo experiments were not blinded during experimentation, although the labels were masked during data analysis.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
