Fig 2: RNaseH2A is regulated by E2F transcription factors.a Schematic diagram showing the E2F-binding sites in the RNaseH2A gene promoter. Red arrows indicate the region containing the PCR primers for ChIP analysis. b ChIP-qPCR analysis of pre-senescent TIG-3 cells using antibodies shown at the bottom. Error bars indicate the mean ± SD of technical triplicates. c RT-qPCR analysis of RNASEH2A and DP1 mRNA expression with siRNA-treated TIG-3 cells. Error bars indicate the mean ± SD of technical triplicates. d Western blot of TIG-3 cells as in c using antibodies shown at right. Alpha-tubulin was used as a loading control. e Left panel: Schematic representation of the reporter construct of human RNASEH2A gene promoter used in the analysis. Firefly luciferase is shown as Luc. Right panel: HEK293T cells were transfected with the indicated reporter plasmids and a Renilla plasmid as an internal control. After 48 h of transfection, luciferase activities were measured. Error bars indicate the mean ± SD of technical triplicates. f Left panel: Schematic representation of the reporter construct of human RNASEH2A gene promoter used in the analysis. The E2F-binding element or its mutant is presented as a black or white rhombus. The firefly luciferase is shown as Luc. Right panel: HEK293T cells were transfected with the indicated reporter plasmids and a Renilla plasmid as an internal control. After 48 h of transfection, luciferase activities were measured. Error bars indicate the mean ± SD of technical triplicates. All data were representative of at least three biological replicates.

Fig 3: RNaseH2A suppresses the expression of SASP factor genes.a Western blot of TIG-3 cells infected with retrovirus encoding flag-tagged RNaseH2A (lanes 3 and 4) or empty vector (lanes 1 and 2). After puromycin selection, cells were infected with retrovirus encoding HRasV12 and subjected to western blot analysis using the antibodies shown at right. Alpha-tubulin was used as a loading control. b In vitro RNase H2-specific activity assay of TIG-3 cells as in a. Error bars indicate the mean ± SD of technical triplicates. c Quantitative PCR analysis of chromosomal DNA in the cytoplasm from TIG-3 cells as in a using primers against chromosomes 3, 10 and 13. Error bars indicate the mean ± SD of technical triplicates. d RT-qPCR analysis of the indicated genes was conducted using RNA extracted from TIG-3 cells as in a. Error bars indicate the mean ± SD of technical triplicates. All data were representative of at least three biological replicates.

Fig 4: RNaseH2A depletion leads to malignant transformation in cancer cells.a Scheme of experiments with HCT116 cells stably expressing TIR1 and RNaseH2A-mAID. Cells were treated with 500 µM auxin to degrade RNaseH2A-mAID for 24 h (b, c, e) or 48 h (d). b Western blot of HCT116 cells as in a using antibodies shown at right. Beta-actin was used as a loading control. c Quantitative PCR analysis of chromosomal DNA in the cytoplasm from HCT116 cells as in a using primers against chromosomes 3 and 10. Error bars indicate the mean ± SD of technical triplicates. d RT-qPCR analysis of the indicated genes was conducted using RNA extracted from HCT116 cells as in a. Error bars indicate the mean ± SD of technical triplicates. e Cell invasion assay using HCT116 cells as in a. Error bars indicate the mean ± SD of technical triplicates. f Top: Schematic representation of mutant alleles of each driver gene. Bottom: Schematic representation model of the evolutionary changes in the gene expression pattern in association with malignant progression phenotypes. g Colon cancer organoids were subjected to immunofluorescence for indicated antibodies. Scale bars indicate 5 µm. White arrows show dsDNA fragments in the cytoplasm. h The percentage of cytoplasmic DNA-positive cells in g. Error bars indicate the mean ± SD of biological triplicates. One-way ANOVA coupled with Dunnett’s multiple comparisons test. i RT-qPCR analysis of the indicated genes was performed using RNA extracted from colon cancer organoids as in g. Error bars indicate the mean ± SD of technical triplicates. All data were representative of at least three biological replicates.

Fig 5: Developmental Defect of Embryos(A) Rnaseh2a+/+, Rnaseh2aRED/RED, Rnaseh2aRED/G37S, Rnaseh2aRED/-, and Rnaseh2a-/- embryos at embryonic days 8.5–11.5. Scale bar, 1 mm.(B) Survival of mouse embryos from crosses of Rnaseh2aRED/+ × Rnaseh2aRED/+ (top) and Rnaseh2aRED/+ × Rnaseh2aG37S/+ (bottom). Expected number of Rnaseh2aRED/RED and Rnaseh2aRED/G37S at Mendelian ratio is shown in right column.(C) Rnaseh2a+/+ and Rnaseh2aG37S/- embryos and neonates. Scale bars, 2 mm (E14.5) and 1 cm (birth).(D) Partial rescue of embryonic lethality by ablation of p53 gene. Images of E10.5 embryos are shown. A morphological rescue of Rnaseh2aRED/RED and Rnaseh2aRED/G37S was observed on p53 background. Rnaseh2aRED/G37S was also rescued on p53+/- background (n = 5), whereas Rnaseh2aRED/RED was not (n = 3). No rescue of Rnaseh2aRED/RED (n = 8) and Rnaseh2aRED/G37S (n = 5) phenotypes was observed on Sting-/- background.