Fig. 1: Replication stress induces lncRNAs associated to replicating chromatin.

a (Top) Mechanism of action of hydroxyurea (HU) RNR: ribonucleotide reductase. (Bottom) Immunoblot analysis HCT116 cells treated with HU 1 mM for 8 h followed by 3 h recovery shows the reversible effect of HU on replication stress markers p-ATR, p-p53 and γH2A.X. Experiments were performed twice with similar results. Source data are provided as a Source Data file. b Schematic of the fractionation protocol applied to isolate chromatin-associated RNAs. c Volcano plot showing the -log10(adjusted p-value) and the log2(fold-change) from the RNA-seq differential expression analysis, comparing the HU-treated vs untreated chromatin fractions. Transcripts with ± 1logFC (FDR < 0.05) are highlighted in blue (-1logFC) and red ( + 1logFC). DESeq2⁵⁴ two-sided, with Benjamini-Hochberg FDR correction, was used to measure differential expression. Volcano plot was drawn using ggplot2 R package. d Distribution of the log2(fold-change) of the HU-upregulated genes in the different cell cycle stages of a synchronized RNA-seq. Data points represent log2-ratio of the number of genes in each replication phase vs the number obtained in the 100 random sets generated. (mean ±2 x standard deviation). Source data are provided as a Source Data file e Temporal expression pattern analysis of the induced lncRNAs in RNA-seq data of cells synchronized in the different phases of the cell cycle. n = 3 (one sample wilcoxon test, no multiple testing corrections). Fold-change is defined as the ratio between the expression in one time point versus the expression in the pool of all the other points. Boxplots represent 25 to 75 percentiles, whiskers are 1.5 x interquartile range (interquartile range = percentile75-percentile25). f Enrichr analysis of the promoters of the differentially expressed genes upon HU treatment, searching against the CHEA Transcription Factor Targets database. g Overlap between the promoter of the upregulated lncRNAs and the binding sites of stress-related transcription factors. The blue squares on the grid show the candidate lncRNAs that have a binding site in their promoter for the given transcription factor.