Abstract
The formation of accessible chromatin around DNA double-strand breaks is essential for their efficient repair1. Although the linker histone H1 is known to facilitate higher-order chromatin compaction2,3, the mechanisms by which H1 modifications regulate chromatin relaxation in response to DNA damage are unclear. Here we show that CTP synthase 1 (CTPS1)-catalysed deamidation of H1 asparagine residues 76 and 77 triggers the sequential acetylation of lysine 75 following DNA damage, and this dual modification of H1 is associated with chromatin opening. Mechanistically, the histone acetyltransferase p300 showed a preference for deamidated H1 as a substrate, establishing H1 deamidation as a prerequisite for subsequent acetylation. Moreover, high expression of CTPS1 was associated with resistance to cancer radiotherapy, in both mouse xenograft models and clinical cohorts. These findings provide new insights into how linker histones regulate dynamic chromatin alterations in the DNA damage response.
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Main
Double-strand breaks (DSBs) are among the most severe forms of DNA damage, as they induce genomic instability. Their efficient repair requires increased chromatin accessibility to facilitate the recruitment of repair factors1. The most well-characterized mechanisms for the rapid opening of chromatin involve acetylation and protein poly(ADP-ribosyl)ation (PARylation), which reduce the charge at the DNA–nucleosome interface4,5,6,7,8,9,10. The mechanisms driving chromatin relaxation during the DNA damage response (DDR), and particularly the roles of various histone modifications in regulating chromatin remodelling, however, remain largely unknown.
The linker histone H1 facilitates the folding of chromatin into a more-compact structure2,3,11,12,13. The disruption of residues within the globular domain of H1, which binds to the DNA entry or exit point of the nucleosome, alters the affinity or mode of binding of H1 to chromatin14,15,16. Given its critical roles in regulating the higher-order structure to create a permissive chromatin environment for DNA damage repair, H1 is intrinsically linked to the DDR. Indeed, depletion of the linker histone H1 impairs DSB repair17,18,19. The functions of H1 are largely dependent of its post-translational modifications (PTMs)3,20,21,22. Although the primary function of H1 is to maintain a compact chromatin structure, it is interesting to note that H1 modifications, such as ubiquitylation, phosphorylation and PARylation, contribute to the DDR mainly by facilitating the recruitment of DSB repair factors, rather than directly affecting chromatin relaxation19,23,24. Thus, fully understanding how H1 modifications influence chromatin accessibility around DSBs is crucial for elucidating the underlying mechanisms.
Deamidation, the removal of an amino group from the side chains of the neutral polar residues asparagine (Asn) and glutamine (Gln), is a unique PTM because it can be either enzymatically or chemically catalysed25. Recent studies have shown that glutamine amidotransferases (GATs), a family of metabolic enzymes that catalyse the removal of nitrogen from glutamine, are capable of deamidating asparagine residues in key signalling molecules that regulate essential biological processes26,27,28. The regulation of B cell lymphoma extra-large deamidation has a critical role in the tumour-specific activity of DNA-damaging antineoplastic agents29,30, suggesting that deamidation might also be a functional PTM affecting the DDR; however, the role of histone deamidation, particularly in response to DNA damage, remains entirely unknown. CTPS1, a member of the GAT family, catalyses de novo CTP synthesis31,32, and because of its role in cell proliferation, its inhibitor is currently undergoing clinical trials as a lymphoma treatment33,34,35,36. Although recent studies have shown that CTPS1 deficiency induces DNA damage37, the detailed mechanism by which CTPS1 contributes to DNA damage repair remains to be determined.
In this study, we found that H1 deamidation at asparagine residues Asn76 and Asn77 is catalysed by CTPS1 in response to DNA damage. This event leads to increased H1 nucleosome decompaction through the acetylation of the nearby Lys75 residue. These data have thus unveiled a potential therapeutic target for certain cancers.
H1(N76D/N77D) regulates DNA damage repair
Cervical cancer is commonly treated with DNA-damaging chemoradiotherapy, but patients often develop resistance to therapy and there is a high rate of recurrence38. We thus used a HeLa cervical cancer cell line as the model in this study. To investigate the role of H1 PTMs in regulating the DDR, particularly during the early stages, we performed two-dimensional gel electrophoresis (2DGE). In parallel analyses, all three abundant H1 isoforms, H1.2, H1.3 and H1.4, showed a reduction in H1 positive charge in response to ionizing radiation (IR) (Fig. 1a and Extended Data Fig. 1a). Given the critical role of chromatin relaxation in the early stages of the DDR, we next investigated the effect of each major H1 variant on chromatin relaxation. Highly condensed chromatin is less readily extracted into high-concentration sodium chloride solutions, and indeed, more H3 was extracted from cells treated with the DNA-damaging compound etoposide (VP16). In addition, the short interfering RNA (siRNA)-mediated knockdown of H1.4 induced the highest level of H3 release among the knocked down H1 variants (Extended Data Fig. 1b and Supplementary Fig. 3a), suggesting that H1.4 exerts the strongest chromatin-condensing effects, which is consistent with previous reports39. We further validated the more relaxed chromatin in H1.4-knockout (H1.4-KO) cells (Extended Data Fig. 1c–e). Therefore, we focused on H1.4 as the representative H1 variant in subsequent experiments and observed a clear shift in H1.4 to the positive pole after IR and VP16 exposure in a dose-dependent and time-dependent manner (Fig. 1b and Extended Data Fig. 1f–h). Next, we evaluated the PTMs of the shifted H1 by mass spectrometry and identified the simultaneous deamidation of Asn76 and Asn77 as the most markedly changed deamidation event following DNA damage (Extended Data Fig. 1i–k and Supplementary Fig. 3b–e). The mutation of H1.4 Asn76 and Asn77 to alanine residues (H1.4-2NA) almost completely abolished the reduction in positive charge on H1.4 during the DDR (Extended Data Fig. 1l).
a, HeLa cells were exposed to 10 Gy IR and released for 2 min. Histones were analysed by immunoblotting after 2DGE (left) or regular SDS–PAGE (middle). The relative quantification of H1.4 forms (wild type (WT) and PTM) in the representative experiment (under the blot) and three independent experiments (right) is shown. Ctrl, control. b, HeLa cells were exposed to the indicated doses of IR and released for 2 min. Histones were analysed by immunoblotting after 2DGE (left) or SDS–PAGE (middle). The relative quantification of H1.4 forms is also shown (right). c, HeLa cells were micro-irradiated and analysed by immunofluorescence for γH2AX (green) and H1(N76D/N77D) (red). Scale bars, 5 μm. d, U2OS-AsiSI-ER-AID cells were treated with or without 4OHT and subjected to CUT&Tag-seq with anti-H1(N76D/N77D). e, Circle plot represents P values obtained when CUT&Tag-seq signals for HR and NHEJ DSBs were compared using an increasing window size. NS, not significant. f, U2OS-AsiSI-ER-AID cells were treated with or without 4OHT and subjected to a ChIP–re-ChIP assay with the indicated antibodies. g,h, Wild-type or H1.4-knockout (KO) HeLa cells stably expressing wild-type or mutant Flag–H1.4 were analysed by a comet assay at the indicated times post-IR (g) or post-VP16 (h). Statistical analyses of the tail moment at the indicated time points are shown. Vec, vector. i,j, Wild-type or H1.4-KO HeLa cells stably expressing wild-type or mutant Flag–H1.4 were analysed by immunofluorescence for RAD51 (i) or 53BP1 (j) foci at the indicated times post-IR. Statistical analyses of foci numbers at the indicated time points are shown. Data represent the mean ± s.d. P values were calculated using Student’s unpaired two-tailed t-tests (a,f–j) or two-sample Wilcoxon test (e). n = 3 samples (a,f), n = 20 or 30 DSBs (e) and n = 50 cells (g–j) from 3 independent experiments.
To directly examine the levels of H1 Asn76 and Asn77 deamidation to aspartic acid (H1(N76D/N77D)), we generated a site-specific antibody targeting H1(N76D/N77D) (Extended Data Fig. 2a–d). We established a quadruple-knockout H1 variant HeLa cell line (H1.1–H1.4-KO; referred to as 4KO) and found that H1(N76D/N77D) signals were much weaker in 4KO cells than in wild-type cells after DNA damage (Extended Data Fig. 2e,f). H1(N76D/N77D) levels increased in response to IR-induced, VP16-induced and laser micro-irradiation-induced DNA damage (Fig. 1c and Extended Data Fig. 2g,h). We further verified the enhanced levels of H1(N76D/N77D) around DSBs using a U2OS-AsiSI-ER-AID cell line, in which site-specific DSBs can be induced by 4-hydroxytamoxifen (4OHT). Cleavage under targets and tagmentation sequencing (CUT&Tag-seq), chromatin immunoprecipitation with quantitative PCR (ChIP–qPCR), and ChIP–re-ChIP analyses all revealed the enrichment of H1(N76D/N77D) at the 80 sensitive damaged sites prone to both homologous recombination and non-homologous end-joining (NHEJ) repair pathways (Fig. 1d–f and Extended Data Fig. 2i). Hydroxyurea-mediated DNA replication stress and long-release period after exposure to ultraviolet radiation C also increased H1(N76D/N77D) levels, probably due to subsequent DSB formation, whereas metabolic or inflammatory stresses, which do not induce DSBs, had no effect (Extended Data Fig. 2j,k). Increased H1(N76D/N77D) is thus a widespread biological phenomenon that occurs in response to DNA damage.
We next explored the role of H1(N76D/N77D) in DNA damage repair. Comet assays showed that compared with H1.4-KO cells, which exhibited severely impaired repair efficiency, knockout cells stably expressing wild-type H1.4 or H1.4 with Asn76 and Asn77 mutated to aspartic acid to mimic deamidation (H1.4-2ND) demonstrated improved DNA repair. By contrast, only a slight recovery in tail moment clearance was observed in cells expressing H1.4-2NA or H1.4-2NR (with mutation of Asn76 and Asn77 to arginine), designed to disrupt deamidation (Fig. 1g,h and Supplementary Fig. 4a–e).
Given that chromatin accessibility facilitates the recruitment of repair factors to DSB sites, we also investigated the foci formation of two typical repair factors: RAD51 (for homologous recombination) and 53BP1 (for NHEJ). As expected, compared with cells expressing wild-type H1.4, H1.4-KO cells expressing an empty vector, H1.4-2NA or H1.4-2NR cells contained reduced numbers of RAD51 and 53BP1 foci, reflecting the decreased efficiency of both repair pathways (Fig. 1i,j and Supplementary Fig. 4f,g). Together, these results confirm that H1 deamidation is essential for DNA damage repair.
CTPS1 deamidates H1 after DNA damage
The human genome encodes 11 GATs25, but only CTPS1 knockdown inhibited the level of H1(N76D/N77D) induced by IR exposure (Extended Data Fig. 3a). We also found that the reintroduction of wild-type CTPS1 efficiently rescued the H1(N76D/N77D) levels in CTPS1-KO cells, whereas enzymatically dead (C399A/H526A/E528A; hereafter termed ED)40 CTPS1 had no effect (Fig. 2a and Supplementary Fig. 5a–c). As Cys399 is mainly responsible for the GAT activity of CTPS1, we also observed a disruption to the H1(N76D/N77D) increase in CTPS1(C399A)-expressing cells (Supplementary Fig. 5d), confirming that the GAT activity of CTPS1 is essential for H1(N76D/N77D).
a, Wild-type or CTPS1-KO HeLa cells stably expressing wild-type or enzymatically dead (ED) Flag–CTPS1 were treated with or without 10 Gy IR. Histones and whole-cell lysates (WCLs) were analysed by immunoblotting. b, GST–H1.4 purified from Escherichia coli was incubated with or without Flag–CTPS1 purified from HEK293T cells. In vitro deamidation reactions were analysed by immunoblotting after 2DGE (left) and SDS–PAGE (right). c,d, Representative cells (c) and quantification (d) of GFP signals in HeLa and HCT116 cells that were transfected with GFP–CTPS1 and exposed to laser micro-irradiation. Scale bars, 5 μm. e, U2OS-AsiSI-ER-AID cells were treated with or without 4OHT and subjected to CUT&Tag-seq with anti-CTPS1. f, Wild-type or CTPS1-KO HeLa cells stably expressing wild-type CTPS1 or CTPS1-ED were analysed by a comet assay at the indicated times after IR. Statistical analyses of the tail moment at 4 h post-IR are shown. 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 then exposed to IR. Cells were analysed by a comet assay at the indicated times after IR. Statistical analyses of the tail moment at 4 h post-IR are shown. Data represent the mean ± s.d. P values were calculated using Student’s unpaired two-tailed t-tests (f,g). n = 8 (d) or n = 50 (f,g) cells from 3 independent experiments.
We next established an inducible CTPS1 degradation system (FKBP–CTPS1) in HeLa cells. Consistent with previous findings, CTPS1 degradation resulted in a diminished IR-induced increase in H1(N76D/N77D) levels (Extended Data Fig. 3b). Validating these findings, we detected an interaction between CTPS1 and H1.4 following IR exposure (Extended Data Fig. 3c,d). In addition, although CTPS1 interacted with all major H1 variants, the most enhanced interaction upon DNA damage was that between CTPS1 and H1.4 (Extended Data Fig. 3e). We also confirmed that histidine-tagged CTPS1 (His–CTPS1) interacted directly with glutathione S-transferase (GST)-tagged H1.4 (GST–H1.4), but not with GST alone or core histones (Extended Data Fig. 3f,g). To confirm the function of CTPS1 as a bona fide deamidase, we performed in vitro deamidation assays. Compared with the no-enzyme control, wild-type CTPS1, but not CTPS1-ED, caused H1.4 to shift towards the positive pole and an increase in H1(N76D/N77D) levels (Fig. 2b and Extended Data Fig. 3h). Finally, we predicted the interaction between the enzymatic pocket of CTPS1 and H1.4 Asn76 and Asn77 (Supplementary Fig. 5e).
Having established a critical role for CTPS1 in H1(N76D/N77D), we hypothesized that this enzyme also contributes to DNA damage repair. We first observed an enrichment of CTPS1 on chromatin following IR or VP16 treatment (Extended Data Fig. 3i,j). We then detected clear CTPS1 enrichment at laser micro-irradiation-induced, AsiSI-induced and FokI-induced DNA damage sites (Fig. 2c–e and Extended Data Fig. 3k–m). This CTPS1 recruitment to DSBs was abolished in 4KO cells but not in H1.4-only knockout cells, which we attributed to the interaction between CTPS1 and other H1 variants (Extended Data Fig. 3n–p and Supplementary Fig. 5f–h). We also found that CTPS1 phosphorylation at Ser575 was induced by DNA damage, a modification essential for CTPS1 recruitment to DSB sites (Supplementary Fig. 5i–k). Overall, we posited that CTPS1 is recruited to DSB sites to mediate H1(N76D/N77D).
We subsequently investigated the role of CTPS1 in DNA damage repair. As expected, CTPS1 knockout or knockdown decreased the repair efficiency of DNA damage induced by IR or VP16. Expression of wild-type CTPS1, but not CTPS1-ED, rescued the inhibition of DNA repair in CTPS1-KO cells (Fig. 2f, Extended Data Fig. 4a and Supplementary Fig. 6a–d). Moreover, expression of wild-type CTPS1, but not CTPS1-ED, rescued the CTPS1-knockdown-induced decrease in repair in both pDR–GFP (for homologous recombination) and pEJ5–GFP (for NHEJ) U2OS reporter cells (Extended Data Fig. 4b and Supplementary Fig. 6e–i). Consistent with this finding, CTPS1-KO cells inhibited the formation of RAD51 and 53BP1 foci following DNA damage, which was rescued by wild-type CTPS1 but not by CTPS1-ED (Extended Data Fig. 4c,d and Supplementary Fig. 6j,k). All these data suggested that the mechanism by which CTPS1 modulates DNA damage repair is enzymatic activity dependent. Although the addition of CTP restored CTP levels in the CTPS1-KO cells, DNA repair efficiency was not restored to basal levels, indicating that the role of CTPS1 in the DDR is independent of CTP synthesis activity (Extended Data Fig. 4e–g and Supplementary Fig. 7a,b).
We further explored the role of H1(N76D/N77D) in the mechanism by which CTPS1 mediates DNA repair. Compared with cells expressing wild-type H1.4, CTPS1-degraded or CTPS1-knockdown cells expressing H1.4-2ND led to an improved DNA repair capacity, whereas H1.4-2NA or H1.4-2NR expression was markedly less effective (Fig. 2g, Extended Data Fig. 4h–k and Supplementary Fig. 7c–g). In addition, both CTPS1 degradation and H1.4-KO in cells led to defects in DNA repair efficiency, with no further defects observed when both CTPS1 and H1.4 were simultaneously disrupted (Supplementary Fig. 8a–f). These findings confirmed that the role of CTPS1 in DNA damage repair is primarily dependent on H1, and that H1(N76D/N77D) is the functional modification site that regulates CTPS1-mediated DNA damage repair.
H1(N76D/N77D) is a prerequisite for H1K75ac
On the basis of the role of H1 as a linker DNA-binding histone, we hypothesized that H1(N76D/N77D) is associated with chromatin relaxation during the DDR. As expected, compared with cells expressing wild-type H1.4-, cells expressing H1.4-2NA or H1.4-2NR showed diminished chromatin relaxation following DNA damage. Although H1.4-2ND expression promoted nucleosome relaxation after DNA damage, it did not induce a more relaxed chromatin state under physiological conditions (Fig. 3a and Extended Data Fig. 5a), indicating that other mechanisms are involved after H1(N76D/N77D).
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.
Our mass spectrometry analysis revealed that the Lys75 residue adjacent to Asn76 is also acetylated (Extended Data Fig. 5b). We therefore generated specific antibodies targeting H1K75 acetylation (H1K75ac) or simultaneous modifications of these three sites (H1K75ac2ND) (Extended Data Fig. 5c–f). Similar to H1(N76D/N77D), the levels of H1K75ac and H1K75ac2ND increased immediately after IR or VP16 exposure (Fig. 3b and Extended Data Fig. 5g). We further confirmed the induction of H1K75ac and H1K75ac2ND in U2OS-AsiSI-ER-AID cells and micro-irradiated cells (Extended Data Fig. 5h–j). Collectively, these data show that H1K75ac and H1K75ac2ND increase in response to DNA damage.
We then explored the relationship between H1(N76D/N77D) and H1K75ac. HeLa cells transfected with wild-type H1.4 or H1.4(K75R) (an arginine substitution mutant of Lys75) showed only minimal differences in H1.4 charge status in response to DNA damage (Extended Data Fig. 6a), suggesting that H1K75ac does not affect H1(N76D/N77D). By contrast, H1K75ac was induced by IR in H1.4 and H1.4-2ND cells, but not in H1.4-2NA or H1.4-2NR cells (Fig. 3c and Extended Data Fig. 6b). Consistent with this finding, CTPS1-KO cells did not show increased levels of H1K75ac or H1K75ac2ND following IR or micro-irradiation (Fig. 3d,e and Extended Data Fig. 6c).
We next examined the influence of H1K75ac and H1K75ac2ND on DNA damage repair efficiency. H1.4-KR, or the triple mutants H1.4-RAA and H1.4-RRR (in which Lys75, Asn76 and Asn77 were substituted to alanine or arginine), compromised DNA break repair compared with wild-type H1.4. By contrast, H1.4-KQ (a glutamine substitution mutant of Lys75) and H1.4-QDD did not result in defective repair (Fig. 3f–i and Supplementary Fig. 9a–f). Together, we conclude that H1(N76D/N77D) is a prerequisite for H1K75ac.
p300 acetylates H1K75 after DNA damage
Next, we sought to identify the histone acetyltransferases (HATs) responsible for H1K75ac. Knockdown of several typical HATs showed that IR-induced H1K75ac was specifically inhibited only when p300 was targeted (Extended Data Fig. 7a); the expression of p300 HAT rescued H1K75ac levels, whereas enzymatically dead (E1399A) p300 HAT had no such effect (Fig. 4a). Next, we confirmed that the interaction between p300 and H1.4 was enhanced in IR-treated cells, and there was a direct interaction between His–H1.4 and the HAT-CH2 domain of p300 in vitro (Fig. 4b,c and Extended Data Fig. 7b,c). We saw a band representing p300 HAT and H1.4 at a higher molecular weight than the band representing p300 HAT alone in native PAGE (Extended Data Fig. 7d). In addition, GFP–p300 HAT colocalized with the FokI–mCherry foci, suggesting that p300 was recruited to DSBs to catalyse H1K75ac (Extended Data Fig. 7e).
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).
Results from an in vitro acetylation assay showed that the catalytic core of p300 was sufficient to acetylate H1K75 (Fig. 4d). However, GST–p300 HAT had no such effect on His–H1.4(K75R) acetylation levels (Extended Data Fig. 7f). We further noted that the inhibited DNA repair efficiency induced by p300 knockdown was improved by H1.4-KQ, but not by H1.4-KR (Extended Data Fig. 7g–j and Supplementary Fig. 10a–h). These data indicate that p300-mediated H1K75ac participates in DNA damage repair.
We next explored the role of H1(N76D/N77D) in p300 recruitment to DNA damage sites. Consistent with the effects of H1(N76D/N77D) inhibition, CTPS1 deficiency abolished the recruitment of p300 to DSBs or damaged chromatin. This defect was rescued upon overexpression of H1.4-2ND, but not H1.4, H1.4-2NA or H1.4-2NR (Fig. 4e and Extended Data Fig. 8a–c). In addition, Flag–H1.4 interacted less with p300 in CTPS1-deficient cells following IR treatment, and Flag–H1.4-2ND expression enhanced the binding of p300 to H1.4, whereas almost no Flag–H1.4-2NA or Flag–H1.4-2NR binding with p300 was detected (Fig. 4f,g and Extended Data Fig. 8d). A GST pull-down assay confirmed an enhanced interaction between GST–p300 HAT and His–H1.4-2ND, which further facilitated the induction of H1K75ac by GST–p300 HAT (Fig. 4h and Extended Data Fig. 8e). Moreover, molecular docking simulations indicated that deamidated H1.4 might be a preferential substrate for p300 HAT due to the closer spatial proximity of the interacting amino acids (Extended Data Fig. 8f).
By contrast, after knocking down p300, we saw similar patterns of CTPS1 chromatin recruitment and H1(N76D/N77D) levels after DNA damage, and also no differences in the binding of CTPS1 to wild-type H1.4, H1.4-KQ or H1.4-KR (Extended Data Fig. 8g–j). We thus concluded that H1K75ac does not affect CTPS1 recruitment to DSB sites or its binding to H1 following DNA damage.
H1K75ac promotes chromatin relaxation
We next investigated whether H1K75ac and H1K75ac2ND are involved in chromatin relaxation. We found that cells expressing H1.4-KR, H1.4-RAA and H1.4-RRR were more resistant to micrococcal nuclease (MNase) digestion and released lower levels of H3 than wild-type H1.4- cells, whereas H1.4-KQ and H1.4-QDD cells showed more relaxed chromatin (Fig. 5a and Extended Data Fig. 9a,b). We also performed the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and found that 4OHT induced a more accessible signal near the AsiSI site (Fig. 5b). In addition, the relaxed regions were highly associated with H1 deamidation and acetylation, as well as with CTPS1-enriched regions (Fig. 5c and Supplementary Fig. 11a). Moreover, H1.4-KO U2OS-AsiSI-ER-AID cells transfected with an H1.4-RAA plasmid exhibited disrupted chromatin relaxation following 4OHT treatment (Fig. 5b). These findings indicate that sequential H1(N76D/N77D) and H1K75ac promote chromatin relaxation during the DDR.
a, Chromatin fractions or WCLs were extracted from H1.4-KO HeLa cells that were transfected with the indicated plasmids, and analysed by an MNase assay (top) or immunoblotting (middle), and the intensity of each lane was quantified (bottom). b, H1.4-KO U2OS-AsiSI-ER-AID cells were transfected with wild-type H1.4 or H1.4-RAA plasmids and treated with or without 4OHT. Cells were subjected to ATAC-seq. Normalized signals of 80 sensitive DSB sites were plotted. c, U2OS-AsiSI-ER-AID cells were treated with or without 4OHT and subjected to ATAC-seq or CUT&Tag-seq with the indicated antibodies. Normalized signals of a representative DSB site is shown. d, H1.4-KO HeLa cells expressing wild-type or mutant Flag–H1.4 were modified to express FKBP–CTPS1, and treated with or without VP16 after being treated with or without dTAGV-1 for 3 h. Chromatin fractions or WCLs were analysed by an MNase assay (top) or immunoblotting (middle). The intensity of each lane was also quantified (bottom). e, The affinity between purified His–H1.4 or His–H1.4-KQ proteins and synthesized DNAs was analysed by an MST assay. f, Wild-type or CTPS1-KO HeLa cells were injected into nude mice, with or without (Ctrl) exposure to IR (n = 6 per group). Growth curves of xenografts are shown. g, Quantification of CTPS1 expression in cervical cancer tissues (n = 58). Blue indicates sensitive patients who survived 5 years after radiotherapy, and red denotes resistant patients who had died within 5 years of radiotherapy. h, Overall survival of patients with survival information in g (n = 48). Blue indicates low CTPS1expression, red denotes high CTPS1 expression. i, Correlation of CTPS1 and H1(N76D/N77D) expression in cervical tumour and para-tumour tissues (n = 57). Data represent the mean ± s.d. (f,g). P values were calculated using Student’s unpaired two-tailed t-tests (f,g), log-rank (Mantel–Cox) test (h) or Pearson r test (i).
We next determined the roles of CTPS1 in H1-mutation-mediated chromatin remodelling. The overexpression of CTPS1, but not CTPS1-ED, rescued the CTPS1-knockdown-induced defects in chromatin relaxation (Extended Data Fig. 9c and Supplementary Fig. 11b). Following CTPS1 downregulation, no improvement in chromatin relaxation was observed in H1.4-expressing, H1.4-2NA-expressing or H1.4-2NR-expressing cells, but the levels of chromatin relaxation were increased in cells expressing H1.4-2ND (Fig. 5d and Extended Data Fig. 9d). We also found less transposase-accessible chromatin around DSB sites in CTPS1-knockdown cells (Extended Data Fig. 9e). Similarly, p300-deficient cells exhibited increased chromatin compaction compared with control cells, which was prevented by p300 HAT overexpression (Supplementary Fig. 11c). Moreover, rescued H1.4-KQ, but not H1.4 or H1.4-KR, expression abolished the increase in chromatin compaction induced by p300 knockdown (Extended Data Fig. 9f,g).
We then demonstrated that the interaction between H1.4 and DNA was approximately six times stronger than that between H1.4-KQ and DNA (Fig. 5e). Lys75, Asn76 and Asn77 form one of the two core binding domains for the interaction of H1.4 with linker DNA. H1.4-QDD showed a more relaxed mode of H1.4 and linker DNA binding, verifying our finding that H1K75ac2ND leads to a lower affinity of acetylated H1 for DNA and therefore chromatin relaxation (Supplementary Fig. 11d). These results demonstrate that sequential CTPS1-mediated H1(N76D/N77D) and p300-mediated H1K75ac facilitate chromatin relaxation upon DNA damage.
We also investigated the importance of PARylation in chromatin relaxation and found no regulatory crosstalk between PARP1 and H1(N76D/N77D) or H1K75ac after DNA damage (Supplementary Fig. 11e–g), indicating that H1 deamidation and PARP1 are two independent pathways during the DDR. Although PARP1 mainly affects core histone compaction41,42, H1 deamidation may function in moderating the affinity between H1 and linker DNA and thus condensing the nucleosome array.
CTPS1 is a radiotherapy sensitizer
Disturbed chromatin relaxation leads to genome instability and chromosome abnormalities, which in turn cause tumour cell death in response to radiotherapy. We found that cells stably expressing H1.4-2NR, H1.4-2NA, H1.4-KR, H1.4-RAA or H1.4-RRR displayed more chromosome breaks after DNA damage (Extended Data Fig. 10a and Supplementary Fig. 12a). Similarly, CTPS1-KO cells exhibited more severe chromosome defects when treated with IR (Extended Data Fig. 10b and Supplementary Fig. 12b), demonstrating that the chromatin relaxation induced by sequential H1(N76D/N77D) and H1K75ac is required for genome stability.
Consistent with these genome stability results, we found that cells stably expressing defective H1.4 mutants showed an increased sensitivity to DNA damage (Extended Data Fig. 10c,d and Supplementary Fig. 12c,d). 4KO cells exhibited slower growth without DNA damage and a greater sensitivity to IR (Supplementary Fig. 12e–g). We also investigated the contribution of CTPS1 to the radiotherapy sensitivity of cells. Although the proliferation of CTPS1-KO cells without DNA damage was inhibited, they showed significantly compromised clonogenic survival following VP16 or IR treatment that was mainly independent of CTP levels (Extended Data Fig. 10e–g and Supplementary Fig. 12h–m).
Next, we explored the role of CTPS1 and H1 deamidation in tumour growth in vivo. Induction of H1(N76D/N77D) was disrupted in H1.4-RAA and H1.4-RRR xenografts, leading to inhibited tumour growth after treatment with IR (Supplementary Fig. 13a–d). Similarly, IR suppressed the growth of CTPS1-KO xenografts to a greater extent than that of wild-type xenografts (Fig. 5f and Extended Data Fig. 10h–j). In addition, a high-CTPS1-expressing cancer cell line exhibited greater resistance to radiotherapy than a low-CTPS1-expressing cancer cell line (Supplementary Fig. 13e–g). These data suggest that CTPS1 has a potential role as a radiosensitizer for cancer therapy.
We finally determined the pathological relevance of CTPS1 in clinical cancer specimens. Of note, a cervical carcinoma tissue microarray isolated from patients before radiotherapy showed that CTPS1 was upregulated in those with subsequent radiotherapy resistance (Fig. 5g and Extended Data Fig. 10k), and overall survival was poorer in patients expressing high CTPS1 levels (Fig. 5h). Another cohort showed a positive correlation between CTPS1 and H1(N76D/N77D) in cervical carcinomas tested after radiotherapy (Fig. 5i and Extended Data Fig. 10l). Analysis of public databases showed that high expression of CTPS1 was also associated with poorer overall survival in patients with DNA damage-based chemotherapy-treated lung and gastric cancer than in the total patient population (Supplementary Fig. 13h). We thus propose that CTPS1 might be a potential target to improve cancer radiotherapy (Extended Data Fig. 10m).
Discussion
Recent spectrometric analyses have revealed an extensive number of H1 PTMs that may be linked to chromatin structure and dynamics11,43. With almost identical sequences, PTMs in the globular domain of the somatic H1 variants are highly conserved, whereas PTMs in the tails of H1 variants vary substantially due to differences in tail sequences11. Therefore, modifications in the globular domain of H1 typically have fundamental roles in various biological processes22,23,44. The disruption of residues within the globular domain affects the affinity or modality of H1 binding to chromatin14,15,16,44. These findings, together with our discovery that the K75Q mutation of H1 decreases chromatin compaction, support the importance of modifications in the H1 globular domain in regulating chromatin relaxation.
Various histone PTMs at DSBs can provide platforms for or directly lead to chromatin relaxation. In the latter case, histone acetylation reduces the number of charge-dependent histone–DNA interactions, thereby inducing chromatin decompaction45. Several histone acetylation sites involved in DNA damage have been reported8,46; however, the mechanisms by which histone acetylation is initiated in the early stages of the DDR have only recently begun to garner attention from researchers6. In the present study, we not only identified an H1 acetylation site (H1K75ac) that facilitates chromatin relaxation in response to DNA damage but also revealed the permissive conditions for this acetylation event. Previous studies have shown that one type of histone modification can be determined by another type of modification on neighbouring residues. For example, H3K4 methylation promotes the binding of the nucleosomal acetyltransferase complex to acetylate histone H3 at Lys14 (refs. 47,48,49,50). On the basis of our results, we propose a similar sequential regulatory model for histone modification crosstalk that is mediated by enhancing the interactions of ‘writers’ with modified histones.
Deamidation rates are controlled by the primary protein sequence, with deamidation occuring increasingly more readily in the sequence order NG > NS > NN > NT > NH51. Accordingly, we found that Asn76 and Asn77 of H1, which are adjacent to Ser78, comprise the only deamidation hotspot in the H1 globular domain sequence. Although the surrounding sequence and cellular environment could accelerate the deamidation rate, non-enzymatic reactions generally vary between 1 and 500 days for asparagine and 500 and 17,900 days for glutamine52. Although these reactions are evidently too slow to function as a stress response, they have been recognized to be a molecular clock of ageing52,53. Compared with non-enzymatic reactions, the protein deamidation catalysed by enzymes is generally rapid and tightly regulated in response to various stimuli54,55,56. In our study, H1 was deamidated by CTPS1 several minutes after DNA damage, suggesting that this enzymatic deamidation is a rapid-response PTM involved in maintaining cellular homeostasis.
Protein deamidation is generally considered to be associated with a loss of biological function54. So far, only a small subset of proteins in prokaryotes and mammalian cells has been shown to gain function after deamidation. Initial studies have shown that the glutamine deamidation of Ras homologue family member A leads to G protein activation57,58. Others have revealed that the deamidation of various signalling molecules facilitates transcriptional activation and immune evasion26,27,28. Here we revealed that CTPS1-catalysed H1(N76D/N77D) may induce a conformational change favourable for p300 binding in response to DNA damage, offering new evidence that deamidation is also an enzyme-catalysed PTM with an essential role in cellular processes. The effects of enzymatic action on restoring asparagine or glutamine from aspartic acid or glutamic acid residues remain to be clarified in future studies.
The role of CTPS1 was initially characterized in the blood system34,36,37, but recent studies have provided evidence for a broader role for CTPS1 across various cancers35,59,60. CTPS1 is reportedly involved in the DNA replication stress caused by pyrimidine deficiency37,60, but the mechanisms by which CTPS1 regulates DNA damage, particularly DSB repair, are unclear. In this study, we discovered that CTPS1 is rapidly recruited to DSB sites, indicating that it functions in a rapid DDR that is independent of the long-term effects of pyrimidine deficiency. In addition, Ser575 phosphorylation of CTPS1 is reported to disrupt its activity40, which indicates a possible dynamic regulation of transitioning from recruitment but lower deamidase activity to higher deamidase activity after recruitment of CTPS1.
In conclusion, our data demonstrate that H1(N76D/N77D) is catalysed by CTPS1 in response to DNA damage. Furthermore, H1(N76D/N77D) is a prerequisite for p300 recruitment to Lys75 for acetylation catalysis and decreased nucleosome compaction. Validation of this regulatory model in mouse models and clinical samples showed that CTPS1 is highly correlated with radiotherapy resistance, suggesting that targeting H1 deamidation is a promising strategy for cancer therapy.
Methods
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.
Data availability
Plasmids and primers used are listed in the Supplementary Information. CUT&Tag-seq and ATAC-seq data generated during this study are available at the Gene Expression Omnibus (GEO) under the accession number GSE288071. Mass spectrometry data are available via ProteomeXchange with the identifier PXD060714. Materials and reagents are available on request. Full immunoblots and gating of flow cytometry are provided as Supplementary Figs. 1 and 2. Source data are provided with this paper.
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Acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (32090030 and 82430096), the National Key Research and Development Program of China (2023YFE0205200), the Shenzhen Municipal Commission of Science and Technology Innovation (JCYJ20220818100015032, JCYJ20220818100016034 and RCYX202170602040047), Shenzhen Medical Research Fund (B2302010) and the Shenzhen University 2035 Program for Excellent Research and Program for Youzuzhikeyan. We thank J. Luo, X. Xu, B. Liu, J. Yuan and M. Tan for their valuable suggestions; G. Legube for providing the AsiSI-ER-U2OS-AID cells; X. Xu for providing PARP1-KO, EJ5-U2OS and DR-U2OS cells; R. Greenberg for providing the U2OS-265 reporter cells; and J. Tamanini of ETediting, UK for English language editing before submission.
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Authors and Affiliations
Contributions
Y.T., J.Z. and W.-G.Z. designed the experiments. Y.T., T.F., W.Z., M.L., W.T. and Y.Z. performed the molecular and biochemical experiments. Y.T., T.F. and Y.S. performed the mouse experiments. Y.T. and T.F. conducted the protein purification experiments. Y.T., T.F., J.Z., M.L. and F.C. performed the cellular irradiation experiments. Q.M. performed the sequencing analyses. M.T. and C.L. performed the sample analyses. S.T., Q.Z. and X.L. provided valuable suggestions for the design of experimental models. Y.T. and W.-G.Z. obtained the funding for this work. Y.T., T.F., J.Z. and W.-G.Z. wrote the manuscript.
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Nature thanks Pinghui Feng, Guohong Li and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
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Extended data figures and tables
Extended Data Fig. 1 DNA damage induces Deamidation of H1.
a, HeLa cells were exposed to 10 Gy IR and released for 2 min. Histones were extracted for 2DGE. b, HeLa cells were transfected with the indicated siRNAs for 48 h and treated with or without VP16. Cell lysates were extracted using 1 M NaCl and analyzed by immunoblotting and CBB (top). WCLs were extracted and analyzed by immunoblotting (bottom). c, WCLs of WT or H1.4-KO HeLa cells were extracted and analyzed by immunoblotting. d, WT or H1.4-KO HeLa cells were treated with or without VP16. Chromatin fractions were extracted and analyzed by MNase sensitivity assay. e, WT or H1.4-KO HeLa cells were treated with or without VP16. Cell lysates were extracted using 1 M NaCl and analyzed by immunoblotting (top) and CBB (bottom). f, HeLa cells were exposed to 10 Gy IR and released for the indicated times. Histones were extracted for immunoblotting after 2DGE (left) or SDS-PAGE (middle). The relative quantification of H1.4 forms (WT and PTM) in the representative experiment is shown (right). g, HeLa cells were exposed to 20 μM VP16 for 2 h. Histones were extracted for 2DGE. h, HeLa cells were exposed to the indicated doses of VP16 for 2 h. Histones were extracted for immunoblotting after 2DGE (left) or regular SDS-PAGE (right). i,j, HeLa cells were exposed to 10 Gy IR or not (Ctrl) and released for 2 min. Histones were extracted and deamidated peptides were identified by mass spectrometry (MS). Ratio (i) and relative quantification (j) of deamidated peptides are shown. k, The identification of N76 and N77 deamidated peptides by MS is shown. The product ions y3 (377.18) and y4 (492.20) indicated the deamidation of the asparagine at the 3rd and 4th position from the C terminus of the peptide. l, H1.4- KO HeLa cells transfected with the indicated FLAG-tagged plasmids (WT or 2NA) for 48 h were exposed to 10 Gy IR or not (Ctrl). Histones were extracted for immunoblotting after 2DGE (left) or regular SDS-PAGE (right). Three independent experiments were performed in (a-j, l).
Extended Data Fig. 2 Deamidation of H1 N76/77 is increased after DNA damage.
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).
Extended Data Fig. 3 CTPS1 deamidates H1 N76/77 in response to DNA damage.
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).
Extended Data Fig. 4 CTPS1 deamidated-H1 N76/77 is required for DNA damage repair.
a, WT or CTPS1-KO HeLa cell lines stably expressing WT or mutant CTPS1 were exposed to VP16 and analyzed by comet assay at the indicated times post-treatment. Statistical analyses of the tail moment at 6 h post treatment are shown. b, pDR-GFP (top) or pEJ5-GFP (bottom) U2OS cells were transfected with the indicated siRNAs and then plasmids, and then infected with a retrovirus expressing I-SceI, before flow cytometric analysis of GFP expression. c,d, WT or CTPS1-KO HeLa cell lines stably expressing WT or mutant CTPS1 were exposed to 10 Gy IR and analyzed by immunofluorescence at the indicated times post-treatment. Statistical analyses of RAD51 (c) and 53BP1 (d) foci numbers at indicated time points post-IR are shown. e, WT or CTPS1-KO HeLa cell lines were treated with or without 200 μM CTP for 2 h. Cells were collected and analyzed by mass spectrometry to determine CTP levels. f,g, WT or CTPS1-KO HeLa cell lines were treated with or without 200 μM CTP for 2 h and exposed to 10 Gy IR and analyzed by comet assays (f) or immunofluorescence to detect 53BP1 foci (g) post-IR. Statistical analyses of the tail moment at 4 h (f) or foci numbers at the 1 h (g) post-IR are shown. h, H1.4-KO HeLa cells stably expressing WT or mutant FLAG-H1.4 were modified to express FKBP-CTPS1, and treated with dTAGV-1 for 3 h. WCLs were extracted and analyzed by immunoblotting. i, pDR-GFP (left) or pEJ5-GFP (right) U2OS cells were transfected with the indicated siRNAs, then with plasmids, and flow cytometric analysis of GFP expression was performed. j,k, H1.4-KO HeLa cells stably expressing WT or mutant FLAG-H1.4 were modified to express FKBP-CTPS1, treated with dTAGV-1 for 3 h, and exposed to 10 Gy IR. Cells were fixed and analyzed by immunofluorescence at the indicated times post-treatment. Statistical analyses of RAD51 (j) and 53BP1 (k) foci numbers at 2 h or 1 h post-treatment are shown. Data represent the means ± s.d. (a-g, i-k). n = 3 samples (b, e, i) or n = 50 cells (a,c,d,f,g,j,k) each group from three independent experiments. P values were calculated using Student unpaired two-tailed t-tests (a-g, i-k). Three independent experiments were performed (a-g).
Extended Data Fig. 5 DNA damage induces H1K75 acetylation.
a, WT or H1.4-KO HeLa cells with stable expression of FLAG-tagged WT or mutant H1.4 were treated with or without VP16. Cell lysates were extracted using 1 M NaCl and analyzed by immunoblotting (top) and CBB (middle). WCLs were analyzed by immunoblotting (bottom). b, HeLa cells were exposed to 10 Gy IR, released for 2 min, and histones were extracted. K75 acetylated and N76/N77 deamidated peptides were simultaneously identified by MS. c, Dot blot analysis of the indicated amounts of peptides using purified anti-H1K75ac (left) or anti-H1K75ac2ND (right) antibody diluted 1:1,000. The sensitivities of the purified antibodies were <1 ng. d, ELISA confirmation that the specificity of the purified H1K75ac (left) and H1K75ac2ND (right) antibodies against the immunogens was >4-fold higher than that of unmodified peptides. e, WT or 4KO HeLa cells were micro-irradiated and analyzed by immunofluorescence at 5 min post-irradiation. f, WT or 4KO HeLa cells were exposed or not to 10 Gy IR and released for 5 min. Histones were extracted for immunoblotting. g, HeLa cells were exposed to the indicated doses of VP16 for 2 h (top) or to 20 μM VP16 for 2 h and released for the indicated times (bottom). Histones were extracted for immunoblotting. h, HeLa cells were analyzed for γH2AX (green) and H1 PTM (red) by immunofluorescence at 5 min post-micro-irradiation. i, U2OS-AsiSI-ER-AID cells were treated with or without 4OHT and subjected to CUT&Tag-seq with anti-H1K75ac or anti-H1K75ac2ND. j, U2OS-AsiSI-ER-AID cells were treated with or without 4OHT and subjected to ChIP-re-ChIP with indicated antibodies. Data represent the means ± s.d. (j). n = 3 samples from three independent experiments (j). P values were calculated using Student unpaired two-tailed t-tests (j). Three independent experiments were performed in (a, c-j). Scale bars, 5 μm (e, h).
Extended Data Fig. 6 H1K75 acetylation does not influence H1N76/77 deamidation.
a, H1.4-KO HeLa cells transfected with the indicated plasmids for 48 h and then exposed to 10 Gy IR. Histones were extracted for immunoblotting after 2DGE (left) or regular SDS-PAGE (right). b, H1.4-KO HeLa cells were transfected with H1.4-WT, -2ND, or -2NR plasmids for 48 h and exposed to 10 Gy IR. WCLs were extracted, immunoprecipitated using anti-FLAG, and analyzed by immunoblotting. c, WT or CTPS1-KO HeLa cells were exposed to 10 Gy IR. Histones or WCLs were extracted and analyzed by immunoblotting. Three independent experiments were performed in (a-c).
Extended Data Fig. 7 p300 acetylates H1K75 in response to DNA damage.
a, HeLa cells were transfected with the indicated siRNAs for 48 h, and exposed or not to 10 Gy IR. Histones and WCLs were extracted and analyzed by immunoblotting. b, HeLa cells were exposed to 10 Gy IR. WCLs were extracted; immunoprecipitated using anti-IgG, anti-H1.4, or anti-p300; and analyzed by immunoblotting. c, Recombinant GST or GST-p300-HAT proteins were incubated with HIS-H1.4 for GST pull-down assays; *indicates specific protein bands identified by CBB staining. d, p300-HAT protein was incubated with or without HIS-H1.4-2ND for 1 h and subjected to native PAGE. Bovine serum albumin (BSA) was loaded as a molecular weight reference. e, U2OS-265 reporter cells were transfected with GFP-p300-HAT plasmid for 48 h, and treated with 4OHT (500 nM) and shield Ι (30 ng/ml) for 6 h. The co-localization of FokI-mCherry (red) and GFP-p300-HAT (green) was analyzed by immunofluorescence. Scale bars, 5 μm. f, GST or GST-p300-HAT was subjected to in vitro acetylation assay using HIS-H1.4-K75R protein purified from E. coli as substrate. g, H1.4-KO HeLa cells stably expressing WT or mutant H1.4 were transfected with the indicated siRNAs, exposed to 10 Gy IR, and analyzed by comet assay at the indicated times post-IR. (h) pDR-GFP (top) or pEJ5-GFP (bottom) U2OS cells were transfected with the indicated siRNAs, followed by plasmids, and flow cytometric analysis of GFP expression was performed. i,j, H1.4-KO HeLa cells stably expressing WT or mutant H1.4 were transfected with the indicated siRNAs, exposed to 10 Gy IR, and analyzed by immunofluorescence at the indicated times post-IR. Quantifications of RAD51 (i) and 53BP1 (j) foci numbers per cell from 50 cells are shown. Data represent the means ± s.d. (g-j). n = 3 samples (h) and n = 50 cells (g, i,j) in each group from three independent experiments (g-j). P values were calculated using Student unpaired two-tailed t-tests (g-j). Three independent experiments were performed in (a-j).
Extended Data Fig. 8 H1N76/77 deamidation promotes p300 binding to chromatin and H1K75 acetylation.
a, WT or CTPS1-KO HeLa cells were transfected with an mCherry-p300-HAT plasmid and exposed to laser micro-irradiation. Images were captured every 10 sec for 5 min (left), and the irradiated path signal intensity in cells was calculated (right). b, WT or CTPS1-KO HeLa cells were exposed to 10 Gy IR and released for the indicated times. Chromatin was extracted and analyzed by immunoblotting. c, WT or CTPS1-KO HeLa cells were transfected with the indicated plasmids and exposed or not to 10 Gy IR. Chromatin was extracted and analyzed by immunoblotting. d, H1.4-KO HeLa cells stably expressing WT or mutant FLAG-H1.4 were modified to express FKBP-CTPS1, treated with dTAGV-1 for 3 h, and exposed to 10 Gy IR, WCLs were extracted, immunoprecipitated using anti-H1.4, and analyzed by immunoblotting. e, Recombinant GST-p300-HAT protein was subjected to in vitro acetylation assay using HIS-H1.4-WT or HIS-H1.4-2ND proteins purified from E. coli as substrates. f, Molecular-docking-predicted 3D structural image of interaction between p300 and H1.4 by GRAMM (gramm.compbio.ku.edu) in H.14-WT (top) and H1.4-2ND (bottom) contexts. Locations of the main interacting amino acids are shown, and residues 75, 76, and 77 are marked. Blue, p300-HAT domain; red, H1.4 globular domain; light red, H1.4 C and N terminals. g, HeLa cells were transfected with indicated siRNAs for 48 h and exposed to 10 Gy IR. Extracted histones or WCLs were analyzed by immunoblotting. h, H1.4-KO HeLa cells were transfected with the indicated siRNAs and then plasmids for 48 h, and exposed to 10 Gy IR. Chromatin and WCLs were extracted and analyzed by immunoblotting. i, H1.4-KO HeLa cells were transfected with siRNA and the indicated plasmids for 48 h and exposed to 10 Gy IR. WCLs were extracted, immunoprecipitated using anti-FLAG, and analyzed by immunoblotting. j, H1.4-KO HeLa cells were transfected with the indicated siRNA and plasmids for 48 h, and exposed to 10 Gy IR. PLAs were performed using anti-FLAG or/and anti-CTPS1. Representative images (left) and quantification of 50 cells each group (right) are shown. Data represent the means ± s.d. (a, j). n = 8 (a) or n = 50 (j) cells in each group from three independent experiments. P values were calculated using Student unpaired two-tailed t-tests (j). Three independent experiments were performed in (a-e, g-i). Scale bars, 5 μm (a, j).
Extended Data Fig. 9 Sequential H1N76/77 deamidation and H1K75 acetylation promotes chromatin relaxation and maintains genome stability.
a, H1.4-KO HeLa cells were transfected with the indicated plasmids for 48 h and treated with or without VP16. Chromatin fractions were extracted and analyzed by MNase sensitivity assay (top). WCLs were extracted and analyzed by immunoblotting (middle). The intensity of each lane was quantified (bottom). b, H1.4-KO HeLa cells stably expressing FLAG-tagged WT or mutant H1.4 were treated with or without VP16. Cell lysates were extracted using 1 M NaCl and analyzed by immunoblotting (top) and CBB (middle). WCLs were extracted and analyzed by immunoblotting (bottom). c, HeLa cells were transfected with the indicated siRNAs and plasmids for 48 h and treated or not with VP16. Cell lysates were extracted using the indicated dose of NaCl buffer and analyzed by immunoblotting (top) and CBB (middle). WCLs were extracted and analyzed by immunoblotting (bottom). d, H1.4-KO HeLa cells were transfected with the indicated siRNAs and plasmids for 48 h and treated with or without VP16. Cell lysates were extracted using 1 M NaCl and analyzed by immunoblotting (top) and CBB (middle). WCLs were extracted and analyzed by immunoblotting (bottom). e, U2OS-AsiSI-ER-AID cells were transfected with the indicated siRNAs for 48 h and then treated with or without 4OHT. Cells were collected and subjected to ATAC-seq, and 80 sensitive DSB sites were plotted. f,g, H1.4-KO HeLa cells were transfected with siRNA control (siCtrl) or sip300, then plasmids for 48 h, and treated with or without VP16. Chromatin fractions were extracted and analyzed by MNase sensitivity assay (f top). WCLs were extracted and analyzed by immunoblotting (f bottom). The intensity of each lane was quantified (g). Three independent experiments were performed in (a-f).
Extended Data Fig. 10 CTPS1 is a potential therapeutic target for cancer.
a, H1.4-KO HeLa cells with stable expression of H1.4 WT or mutants were exposed or not to 3 Gy IR and subjected to metaphase assay. Chromosomes were counterstained with Giemsa and the aberrations were quantified. b, WT or CTPS1-KO HeLa cells were exposed or not to IR and subjected to metaphase assay. Chromosomes were counterstained with Giemsa and the aberrations were quantified. c,d, WT or H1.4-KO HeLa cell lines stably expressing WT or mutant H1.4 were subjected to colony formation assay after IR (c) or VP16 (d) exposure. Statistical analyses of survival rates at the last dose are shown. e,f, WT or two CTPS1-KO HeLa cell lines were subjected to colony formation assay after VP16 (e) or IR (f) exposure and the survival rate was quantified. g, WT or CTPS1-KO HeLa cell lines were treated with or without 200 μM CTP for 2 h and exposed to IR. Cells were sub-seeded for colony formation assay and the survival rate was quantified. h-j, WT or CTPS1-KO HeLa cells were injected into nude mice, followed by treatment with or without IR (n = 6 per group). Tumor images (h left) and tumor weights (h right) of xenografts are shown. Immunoblots of extracted histones (i), and representative immunohistochemistry and TUNEL staining images of xenografts (j) are shown. Scale bars, 50 μm. k, Representative immunohistochemistry images of CTPS1 expression in cervical cancer tissues isolated from patients before exposure to radiotherapy. Sensitive, patients who were survived after 5 years post radiotherapy; Resistant, patients who were dead within 5 years post radiotherapy. l, Representative western blot images of CTPS1 and H1N76/77D expression in 8 cervical cancer tissues isolated from patients after exposure to radiotherapy. m, Model of H1N76/77D-mediated regulation of chromatin structure in the context of DNA damage. Deamidation of H1N76/77 catalyzed by CTPS1 is prerequisite for a sequential binding of histone acetyltransferase p300 to the reaction site of H1K75. This specific H1K75 acetylation is well associated with an opening structure of damaged chromatin and is therefore beneficial for DNA damage repair factors loading onto the DNA damage sites. The working model was created using BioRender (https://biorender.com). Data represent the means ± s.d. (a-c, e-h) or means (d). n = 5 fields (a,b), n = 3 samples (c, g), n = 2 (d) samples, or n = 4 samples (e,f) from three independent experiments. P values were calculated using Student unpaired two-tailed t-tests (c-h). Scale bars, 50 μm (j).
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Tian, Y., Feng, T., Zhang, J. et al. Histone H1 deamidation facilitates chromatin relaxation for DNA repair. Nature 641, 779–787 (2025). https://doi.org/10.1038/s41586-025-08835-0
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DOI: https://doi.org/10.1038/s41586-025-08835-0
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