Journal: bioRxiv
Article Title: LSH-mediated resolution of R-loops mitigates transcription-replication conflicts to preserve genomic stability in prostate cancer cells
doi: 10.1101/2025.06.04.657812
Figure Lengend Snippet: A The Venn diagram illustrates the number of overlapping genes (n = 471) that are downregulated in LSH KO cells (KO vs. WT, red) and restored in KO+RNH1 cells (blue). Data are representative of n = 3 biologically independent experiments. B Pathway analysis of the overlapping genes was conducted, with a primary focus on cancer signaling pathways, transcriptional activity, and cellular biological processes. Data are representative of n = 3 biologically independent experiments. C Heat map displaying the relative expression levels of overlapping genes in WT, KO, and KO+RNH1 PC3 cells, as outlined in the pathway analysis from panel B. Data are representative of n = 3 biologically independent experiments. D The average R-loop CUT&Tag signals over 6 kb regions centered on the TSS of overlapping genes were analyzed in WT, KO, and KO+RNH1 PC3 cells. Samples treated with the RNH1 enzyme served as a negative control to confirm the specificity of the detected R-loop signals. Data are representative of n = 3 biologically independent experiments. TSS denotes transcription start site. E Co-IP experiments were performed in PC3 cells to evaluate the interaction between LSH and MYC as well as E2F3 proteins. Whole-cell lysates were collected and subjected to immunoprecipitation using either an anti-LSH antibody or an IgG control. Immunoblotting was subsequently carried out with anti-MYC and anti-E2F3 antibodies. Input samples are indicated, and the data represent n = 3 biologically independent experiments. F Enrichment plots for MYC and E2F targets were generated to compare WT and KO cells and further validated by the comparison of KO cells with KO+RNH1 cells. Data are representative of n = 3 biologically independent experiments. G The heat map illustrates the relative expression levels of five selected MYC and E2F target genes among the overlapping genes in WT, KO, and KO+RNH1 PC3 cells. Data are representative of n = 3 biologically independent experiments. H Genome browser snapshots of CUT&Tag-seq data depict R-loop accumulation at the selected MYC target genes, as illustrated in panel G, for WT, KO, and KO+RNH1 PC3 cells. I ChIP–qPCR analysis of γ-H2AX at the selected MYC target genes, as illustrated in panel G, for WT, KO, and KO+RNH1 PC3 cells. Values were normalized to the WT group and presented as a bar graph with mean ± s.d. (n = 3 biologically independent experiments). **p < 0.01; ***p < 0.001; ****p < 0.0001, as determined by the One-way ANOVA with Tukey’s multiple comparison test. J ChIP–qPCR analysis of FANCD2 at the selected MYC target genes, as shown in panel G, for WT and KO PC3 cells. Data are presented as a bar graph with mean ± s.d. (n = 3 biologically independent experiments). *p < 0.05; **p < 0.01; ***p < 0.001, as determined by the two- tailed Mann–Whitney test. K ChIP–qPCR analysis of RNAPIIS2P at the promoter and gene body regions of the selected MYC target genes, as shown in panel G, for WT, KO, and KO+RNH1 PC3 cells. Values were normalized to the WT group and presented as a bar graph with mean ± s.d. (n = 3 biologically independent experiments). **p < 0.01; ***p < 0.001; ****p < 0.0001, as determined by the One-way ANOVA with Tukey’s multiple comparison test. L Genome browser snapshots of CUT&Tag-seq data depict R-loop accumulation at the selected E2F target genes, as illustrated in panel G, for WT, KO, and KO+RNH1 PC3 cells. M ChIP–qPCR analysis of γ-H2AX at the selected E2F target genes, as illustrated in panel G, for WT, KO, and KO+RNH1 PC3 cells. Values were normalized to the WT group and presented as a bar graph with mean ± s.d. (n = 3 biologically independent experiments). **p < 0.01; ***p < 0.001; ****p < 0.0001, as determined by the One-way ANOVA with Tukey’s multiple comparison test. N ChIP–qPCR analysis of FANCD2 at the selected E2F target genes, as shown in panel G, for WT and KO PC3 cells. Data are presented as a bar graph with mean ±s.d. (n = 3 biologically independent experiments). **p < 0.01; ***p < 0.001, as determined by the two-tailed Mann–Whitney test. O ChIP–qPCR analysis of RNAPIIS2P at the promoter and gene body regions of the selected E2F target genes, as shown in panel G, for WT, KO, and KO+RNH1 PC3 cells. Values were normalized to the WT group and presented as a bar graph with mean ± s.d. (n = 3 biologically independent experiments). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, as determined by the One-way ANOVA with Tukey’s multiple comparison test. Source data are provided as a Source Data file.
Article Snippet: The cell lines utilized in this study included the androgen-independent prostate cancer cell line PC3 (ATCC), the androgen-dependent prostate cancer cell line LNCaP (ATCC), and the human embryonic kidney epithelial cell line HEK293T (ATCC).
Techniques: Protein-Protein interactions, Activity Assay, Expressing, Negative Control, Co-Immunoprecipitation Assay, Immunoprecipitation, Control, Western Blot, Generated, Comparison, ChIP-qPCR, Two Tailed Test, MANN-WHITNEY
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