Fig 2: SIRT1 and BMI1 are required for the transcriptional repression of genes near the stalled replication fork. (A–E) Relative mRNA levels of the indicated genes were examined by RT-qPCR in U2OS (A), 293T (B), HCT116 (C), SIRT1-KO (D) and BMI1-KO (E) cells treated with DMSO or doxorubicin. (F and G) H2AK119cr (F) and H2AK119ub (G) ChIP assays of selected genes were performed in U2OS cells treated with DMSO or doxorubicin. The ChIP value in the DMSO-treated cells was set as 1 for normalization. (H) RNA Pol II ChIP assays of the selected genes were performed in U2OS cells, SIRT1-KO, and BMI1-KO cells treated with DMSO or doxorubicin. The ChIP value in the DMSO-treated cells was set as 1 for normalization. In all panels, data represent the means ± SD of three independent experiments. The P-value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001., n.s., not significant; Student’s t-test.

Fig 3: Depletion of SIRT1 or BMI1 during replication stress increases TRCs. (A) Depletion of SIRT1 or BMI1 increases replication stress. (Top) (A) Schematic showing how replication speed was measured using DNA fiber analysis. U2OS cells were pre-treated with 0.2 mM hydroxyurea for 4 h then sequentially pulse-labeled with CldU (green) and IdU (red) for 20 min each in the presence of hydroxyurea. Representative DNA fibers show an elongating fork and a stalled fork (bottom, left). The length of the IdU (red) fibers in the elongating fork was measured using ImageJ software and used to calculate the speed of the replication fork. The scatterplot shows the quantification of the speed of replication fork from at least 200 fibers (bottom right). (B) Recruitment of SIRT1 (top) and BMI1 (bottom) to stalled forks was analyzed by PLA assays. U2OS cells were treated with or without (no) 2 mM hydroxyurea for 4 h; then, SIRT1/EdU PLA assays and BMI1/EdU PLA assays were performed as previously described. Left, representative images. Right, quantification of the average number of PLA foci per nucleus. (C) Parental (WT), SIRT1-KO, and BMI1-KO U2OS cells expressing vector control (ctrl) or the indicated shRNA were treated with 0.5 mM hydroxyurea for 12 h, then PLA between PCNA and RNA pol II was performed. Left, representative images of PLA foci (green). Right, the scatterplot shows the quantification of the PLA signal per nucleus from at least 100 cells. (D and E) The cells used and treated as described in panel (C) were subjected to immunostaining. Left, representative immunostaining images. Right, quantification of S9.6 nuclear signal intensity following nucleolar signal removal. In all panels, data are plotted from three independent experiments with means ± SD. The P-values are indicated as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001., n.s., not significant; Mann–Whitney test; scale bar, 10 μm.

Fig 4: H2AK119cr and H2AK119ub are involved in resolving replication stress-induced TRCs. (A) Chromatin fractions were extracted from cells expressing FLAG-H2A or mutant FLAG-H2K119R and analyzed by western blot using the indicated antibodies. (B) Cells expressing FLAG-H2A or FLAG-H2K119R were treated with or without 1 mM hydroxyurea for 4 h. The chromatin fractions were extracted and subjected to immunoprecipitation with M2 beads followed by immunoblotting. (C) U2OS cells expressing FLAG-H2A or mutant FLAG-H2AK119R were treated with or without (no) 0.5 mM hydroxyurea for 12 h, then subjected to a PCNA/RNA Pol II PLA assay. Representative images of PLA foci and the quantification of PLA signals per nucleus are shown. (D) Cells used and treated as described in panel C were subjected to immunostaining using an S9.6 antibody. Representative images and the quantification of the S9.6 nuclear signal intensity following nucleolar signal removal are shown. (E) Top, chromatin fractions were extracted from SIRT1-KO cells expressing FLAG-H2A2KR or FLAG-H2A2KR-ub fusion constructs and analyzed by western blot. Bottom, a schematic representation of FLAG-H2A2KR and FLAG-H2K1192KR-ub fusion constructs. In both the H2A2KR and fusion constructs, H2A carried a lysine-to-arginine mutation at lysine 118 (K118) and lysine 119 (K119). In the fusion constructs, seven lysines in the ubiquitin were mutated to arginine. (F and G) SIRT1-KO cells expressing FLAG-H2A2KR or FLAG-H2A2KR fusion were subjected to a PCNA/RNA Pol II PLA assay (F), as described in panel C, or immunostaining (G), as described in panel D. In panels C, D, F and G, the data plotted are means ± SD from three independent experiments. The P-values are indicated as ***P < 0.001, ****P < 0.0001. Mann–Whitney test; scale bar: 10 μm.

Fig 5: SIRT1 promotes decrotonylation and subsequent ubiquitination of H2AK119 during replication stress. (A and B) 293T cells were incubated with or without nicotinamide (NAM, 10 mM for 24 h, A) or trichostatin A (TSA, 1 μM for 24 h, B) then treated with 1 mM hydroxyurea for 4 h. After drug treatment, histones were extracted and subjected to western blot analysis using the indicated antibodies. γH2AX was used as a marker of DNA damage induction. The blue staining in the bottom panel shows the loading control. (C) Whole-cell lysates and histones were prepared from 293T cells transfected with the indicated construct and immunoblotted with the indicated antibodies. (D) Histones from wild-type (WT) or SIRT1-KO cells were analyzed by immunoblotting with the indicated antibodies. (E) In vitro decrotonylation assay using extracted histones from SIRT1-KO cells and purified SIRT1 enzyme. The reaction products were analyzed by western blotting with the indicated antibodies. (F) In vitro decrotonylation assay with crotonylated H2A peptides (K119cr) or uncrotonylated H2A peptides (K119) and increasing amounts of SIRT1 or buffer control. The reaction products were spotted on a nitrocellulose membrane and immunoblotted with an H2AK119cr antibody. (G) Histones from WT and BMI1-KO cells were analyzed by immunoblotting with the indicated antibodies. (H and I) SIRT1-KO cells (H) and BMI1-KO (I) cells were pre-treated with the indicated drugs. After drug treatment, histones were extracted and subjected to western blot analysis using the indicated antibodies. H2AK119cr and H2AK119ub blots were quantified using ImageJ software, and the normalized data are shown at the bottom of the blot.