Fig 2: Global histone hyperacetylation improves early G1 heterochromatin MCM loading. (A) Representative immunoblot of histone H3 acetylation in untreated and TSA-treated asynchronously proliferating RPE1-hTert cells (expressing PCNA-mTurquoise and DHB-mCherry). Cells were treated with the indicated concentrations of TSA for 3 h. Quantification of total loaded MCM3 concentration (B), and the ratio of loaded MCM3 concentration in heterochromatin to euchromatin (C) in early G1 cells that had been treated with 300 nM TSA for 3 h. Three replicates are shown. One-way ANOVA test and n (number of cells) is indicated in the figure. ****P = 0.0001.

Fig 3: Impact of DNA damage and CREB S111 phosphorylation on coactivator recruitment and DNA binding activity. (A) ChIP analysis of CREB, CBP and CRTC2 over the CREM promoter in CREB+/+ and CREBS111A MEFs. Cells were treated with Fsk for 90 min, irradiated with 20 Gy IR for 120 min (20 Gy) or treated with 20 Gy IR 30 min prior to Fsk (20 Gy-Fsk) and processed for ChIP-qPCR using the indicated antibodies. Y-axis represents the CREM promoter occupancy. Each bar represents averaged results, n = 3. Error bars indicate SEM. *P < 0.05 (Student's t-test). (B) DNA damage inhibits CREB–CRTC2–DNA ternary complex formation in an S111 phosphorylation-dependent manner. HEK 293T cells transfected with CREBWT or CREB3A were treated with 2 nM CLM for 1 h and cell lysates incubated with GST-CRTC2CBD and either CRE-containing or scrambled oligonucleotides in the presence of GSH-agarose beads. Immobilized CREB–CRTC2–DNA ternary complexes were washed under indicated buffer conditions analyzed by western blotting with a-CREB and a-GST antibodies. (C) Immortalized CREB+/+ and CREBS111A MEFs were treated with Fsk for 90 min, treated with 2 nM CLM for 120 min (CLM), or treated with CLM for 30 min prior to Fsk (CLM + Fsk). CREB–CRE complexes were analyzed by electrophoretic mobility shift assay (EMSA). (D) DNA-binding activity was analyzed by EMSA using cell lysates prepared from HEK 293T cells transfected with empty vector, or plasmids encoding CREBWT, CREB3A (100A, 111A and 121A), CREBQ112D or DNA-binding defective KCREB. The transfected cells were irradiated with 50 Gy IR for 1 h and CREB expression analyzed by immunoblotting. (E) CRE binding activity was measured by densitometric analysis. *P < 0.05 (Student's t-test). (F) DNA damage caused dissociation of CREB from bulk chromatin. HeLa cells were irradiated with 10 Gy IR, collected at the indicated times, and fractionated into soluble (Sol) or chromatin (Chr) fractions prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresisand immunoblotting with a-CREB or a-MCM3 antibodies. (G) CREB chromatin dissociation is ATM dependent. HeLa cells were pretreated with DMSO or ATM inhibitor (KU-55933) for 30 min, and then irradiated with 10 Gy IR for 1 h. Soluble (Sol) or chromatin (Chr) fractions were analyzed using a-CREB, a-HSP90, a-MCM3 or a-TDP-43 antibodies. (H) The S111A mutation inhibits IR-dependent CREB chromatin eviction. Chromatin fractionation of CREB+/+ and CREBS111A MEFs was carried out 1 h after irradiation of 10 Gy IR.

Fig 4: Cancer helper role of E2F1 and MCM7. a E2F1 and MCM7 expression in EACs with amplification (n = 11 samples each) compared to copy number neutral EACs (n = 81 samples each). Significance was assessed by Wilcoxon rank-sum test. b E2F1 and MCM7 mRNA expression in FLO-1 cells. Lower and upper hinges and middle line of boxplots correspond to 25th, 75th, and 50th percentiles. Upper and lower whiskers extend less than 1.5 times the interquartile range. c Proliferation of FLO-1 cells overexpressing E2F1 or MCM7 compared to control. d. EdU (5-ethynyl-2'-deoxyuridine) incorporation by flow cytometry in E2F1 overexpressing cells compared to control. Cells were separated into G1, S and G2 phases. S phase cells were subdivided into 4 gates (S1-S4, Supplementary Fig. 9). Differences in EdU geometric mean fluorescence intensity were assessed by Mann-Whitney U test. MCM complex loading onto chromatin in MCM7 overexpressing or control cells through MCM7 e or MCM3 f staining. Cells were pulsed with EdU and chromatin was fractioned before staining for MCM7 or MCM3 to detect chromatin-bound MCM complex. Cells were separated into cell cycle phases using EdU and DAPI intensity. MCM7 or MCM3 fluorescence intensity during S phase illustrates MCM complex unloading from chromatin. Differences in geometric mean fluorescence intensity of MCM staining were assessed using Mann-Whitney U test. For (d), (e), (f) one representative of n = 3 biological replicates is shown. Corresponding pseudocolour plots are in Supplementary Fig. 9. g MCM7 mRNA expression in MFD-1 and FLO-1 cells. h qRT-PCR MCM7 expression in MFD-1 cells after transduction with a MCM7-shRNA inducible lentiviral vector. Expression was assessed with or without 96 h doxycycline treatment. i Proliferation curve of MFD-1 cells with or without doxycycline-induced MCM7 knockdown. For all qRT-PCR experiments, expression was relativised to ß-2-microglobulin and normalised to FLO-1 cells. N = 2 biological replicates were performed, each in technical triplicate. For all proliferation assays, n = 2 biological replicates were performed, each with four technical replicates. Proliferation was assessed every 24 h and normalised to time zero. Mean values at 72 h were compared by two-tailed Student’s t-test. Error bars show standard deviation. Source data are available in the Source Data file

Fig 5: Preferential heterochromatin underlicensing in shortened G1 cells. (A) Ectopic cyclin E expression was induced by treatment with 15 ng/ml dox for 7 h prior to analysis by immunoblotting. (B) G1 length of cells expressing PCNA-mTurquoise, DHB-mCherry and dox-inducible cyclin E in (A) measured by live-cell imaging of the PCNA S phase reporter; t-test, n (number of cells) = 40 for each group. (C) CDK1/2 activity relative to physical age in control (gray dots) or cyclin E-expressing (red triangles) G1 phase cells treated as in (B). (D) Loaded MCM3 concentration relative to physical age in control (gray dots) or cyclin E-expressing (red triangles) G1 cells as in (B). (E) Loaded MCM concentration in heterochromatin and euchromatin in G1 subphases in control or cyclin E-expressing cells. G1 subphases for (E) and (F) were defined for control cells as in Figure 1 and for cyclin E-overproducing cells by physical age as indicated in (C). Boxplots show median and interquartile ranges, one-way ANOVA, Tukey post-hoc test and n (number of cells) is indicated in the figure. (F) Quantification of the ratio of loaded MCM concentration in heterochromatin to euchromatin in control or cyclin E-expressing G1 cells. Violin plots indicate the median and interquartile ranges, one-way ANOVA, Tukey post-hoc test and n (number of cells) is indicated in the figure. In all panels, P-value ranges are indicated as *P = 0.05, **P = 0.01, ***P = 0.001 and ****P = 0.0001.