Fig 2: Linker histone H1.2 dissociation and destabilization are required for DNA repair and cell survival. a, b Wild-type and H1.2 KO (1# and 2#) HeLa cells were analyzed by comet and colony formation assays. The tail moment of wild-type cells at 10 min post treatment was normalized to 1. The data represent the mean ± SD. c Wild-type and H1.2 KO (1# and 2#) DR-GFP U2OS cells were analyzed by DR-GFP assay. The data represent the mean ± SD. d Wild-type and H1.2 KO pEJ5-GFP U2OS cells were analyzed by EJ5-GFP assay. The data represent the mean ± SD. e, f Wild-type, H1.2 KO (1#), two ATM KO (1# and 3#) and two ATM/H1.2 double KO (5# and 6#) HeLa cells were analyzed by comet and colony formation assays. The tail moment of wild type cells at 12 h post treatment was normalized to 1. The data represent the mean ± SD. g, h Wild-type, H1.2 KO (1#), H1.2 KO (1#) with reintroduced wild type or S188A mutated H1.2, and ATM KO (1#) HeLa cells were analyzed by comet and colony formation assays. ATM KO (1#) HeLa cells with reintroduced wild-type or S188A mutated H1.2 were also analyzed by colony formation assay. The tail moment of wild-type cells at 10 min post treatment was normalized to 1. The data represent the mean ± SD. i A schematic model for the dynamic regulation of ATM by H1.2. In the absence of DNA damage, H1.2 binds to the chromatin and blocks the interactions between ATM and MRN to prevent the recruitment and activation of ATM. Upon DNA damage, PARP1 is activated to PARylate and displace H1.2 from chromatin, whereby ATM is permitted to be recruited and activated by MRN and DNA breaks. Activated ATM, which is amplified by an ATM-MDC1-MRN positive feedback loop, drives the DNA damage response through phosphorylation of a wide spectrum of substrates, including H2AX

Fig 3: SIRT2, TDP-43 and R-loops function to protect cells from infection induced DNA damage.Immunoblot detection of γH2Ax and total H2Ax from whole cell lysates of HeLa cells transfected with stated siRNAs and left uninfected (-) or infected (+) for (A) 6 hours or (B) 24 hours. Quantified band intensities of γH2Ax levels are present in graphs (right). Results are expressed as intensity of actin normalised γH2Ax bands relative to actin normalised total H2Ax. Graph shows the mean ± SEM from at least three independent experiments. Statistical significance was determined by one-way ANOVA with FDR Benjamini-Hochberg (BH) correction for multiple comparisons (ns = not significant, ** = p <0.01, **** = p <0.0001). (C) HeLa cells expressing either mCherry or mCherry-RNaseH1 were infected for 24 hours, immunoblot analyses and quantification of γH2Ax (right) performed as stated for (A) and (B) *denotes non-specific band. Graph shows the mean ± SEM from five independent experiments. Statistical significance was determined by Two-tailed Unpaired t test (*** = p <0.001).

Fig 4: CR-CSphCs resistance to NORA234 is mediated by increased activity of CHK1-mediated DDR(A) Cell cycle analysis in CR-CSphCs treated with a vehicle or NORA234 (red bars), for 24 h. The data show percentage of cell number in G0–G1, S, and G2–M phases. Data are expressed as mean ± SD of three independent experiments performed with cells isolated from 4 different patients with CRC (CR-CSphCs #3, #9, #21, #R4). (Lower panels) Representative cell cycle analysis of CR-CSphCs (#R3) treated with vehicle or NORA234, for 24 h.(B) Representative immunoblot analysis and quantification of DNA damage (y-H2AX), apoptosis (cleaved PARP), and DDR (RAD51) markers expression in CR-CSphCs treated with vehicle or NORA234 at the indicated time points. β-actin was used as loading control. Data are presented as mean ± S.D. of 2 independent experiments performed with cells isolated from 4 different patients with CRC (CR-CSphCs #3, #9, #21, #R4).(C) Heatmap of DNA damage – ATM/ATR regulation of G2-M-checkpoint-related genes (2−ΔΔCt expression values) in CR-CSphCs treated as in (B). Data are presented as normalized expression values of cells isolated from 4 different CRC patients (CR-CSphCs #3, #9, #21, #R4). Black arrow heads indicate genes upregulated upon 24 h of treatment with NORA234, which values are restored to basal levels after 48 h of drug washout.(D) Representative immunocytochemistry analysis of nuclear p-CHK1, in CR-CSphCs (#21) treated with a vehicle, 5-FU in combination with oxaliplatin, or NORA234, at 24 h. Scale bars, 100 μm.(E) Immunoblot analysis of p-CHK1 and CHK1 in wt CR-CSphCs (CSphC #21, #24, and #33) treated as indicated for 24 h. β-actin was used as a loading control.(F) Immunoblot and its relative OD ratio analysis of p-CHK1 and CHK1 in wt, Braf, Kras, or chemoresistant CR-CSphCs. β-actin was used as a loading control. Data are presented as mean ± S.D. of 2 independent experiments performed with cells isolated from 4 different patients with CRC (CR-CSphCs # 21, # 3, # 9, #R4).(G) Box and violin plot of p-CHK1 positivity, evaluated by flow cytometry, in wt (CR-CSphCs #7, #14, #24, #35, #37, #58), Braf (CR-CSphCs #1, #2, #3, #4), Kras (CR-CSphCs #8, #9, #10, #11, #12, #15, #16, #20, #22, #57, #59), or chemoresistant CR-CSphCs (CR-CSphCs #R2, #R3, #R4). Data are expressed as mean ± SD of three independent experiments. Statistical significance between 2 groups was determined by unpaired Student's t-test (2-tailed). ∗p ≤ 0.05; ∗∗p ≤ 0.01.See also Figure S2.

Fig 5: Oxidative stress reduces BNC2 expression in vitro and in vivo. (a) Western blot detection of BNC2 protein levels in NP40-insoluble extracts from COV318 and COV504 cells treated with increasing amounts of H2O2 (0.06, 0.125 and 0.25 mM) at 3 or 24 h post-treatment. Total H2AX was used as loading control. Phospho-H2AX (pH2AX) was detected to check effectiveness of H2O2 treatment. (b) qRT-PCR analysis of BNC2 expression in COV318 and OVCAR4 at 5 h after H2O2 treatment. Bars indicate mean±S.E. from two independent experiments. (c) Western blot detection of BNC2 protein levels in NP40-insoluble extracts from COV318 cells treated with 0.06 mM H2O2 and collected 1, 6 and 24 h post-treatment. Total H2AX was used as loading control. pH2AX was detected to check effectiveness of H2O2 treatment. (d) qRT-PCR analysis of BNC2 expression in COV318 at 1, 6 and 24 h after 0.06 mM H2O2 treatment. Bars indicate mean±S.E. from two independent experiments. (e) ChIP for H3K4me3 enrichment (measured by qRT-PCR) of three regulatory elements of BNC2 locus in COV318 treated with 0.06 mM H2O2 at different time points. Bars indicate mean±S.E. from two independent experiments. (f) Western blot detection of BNC2 protein levels in NP40-insoluble extracts from COV318 cells treated with 0.06 mM H2O2 and collected 1 week post-treatment. Total H2AX was used as loading control. pH2AX was detected to check effectiveness of H2O2 treatment. (g) qRT-PCR analysis of BNC2 expression in COV318 at 1 week after 0.06 mM H2O2 treatment. Bars indicate mean±S.E. from two independent experiments. (h) ChIP for H3K4me3 enrichment (left panel) and H3K27me3 enrichment (right panel) (measured by qRT-PCR) of three regulatory elements of BNC2 locus in COV318 at 1 week after 0.06 mM H2O2 treatment. Bars indicate mean±S.E. from two independent experiments. (i) Left panel: Western blot detection of BNC2 protein levels in NP40-insoluble extracts from prepubertal mice oviducts treated or not with hCG 16 h earlier. COV318 was used as positive control for BNC2 expression. Right panel: Densitometric analysis of BNC2 western blot bands in oviducts from hCG treated (n=10) and untreated (n=10) prepubertal mice as described above. Scatter dot plot bars indicate mean±S.E. from two independent experiments, including the one presented in the left panel. *P<0.05