Figure 4
R-loops form in early-replicating regions with TSS-proximal DSBs, but RDC-genes are not prone to R-loop formation in NSPCs undergoing mild replication stress. (A) NSPC genes with TSS-proximal (± 2 kb) breakpoint junctions ("A", n = 382) show a significantly higher R-loop peak density than RDC-genes with R-loop peaks ("B", n = 23) overall. Whiskers show minimum and maximum values; top and bottom edges of boxplots correspond to 25th and 75th percentiles, respectively; horizontal lines indicate the median. P value was determined by the Mann–Whitney U test. (B) Left, Transcriptional activity of class A (n = 382) and transcription-matched gene set A' (n = 367); Right, Box-and-whisker plots of R-loop peak density in the two groups. Plot details are as in (A). (C) Violin plots showing the frequency distribution of replication timing ratios of all R-loop peaks (n = 22,132), Group 1–3 RDCs (n = 113), and the set of 27 RDC-genes in NSPCs. Median (blue line) and quartile lines (black) are shown. P values were determined by one-way ANOVA with Tukey’s post hoc correction. (D) Parts-of-whole graph showing fraction of RDC-gene HTGTS junctions that fall within two kb of an R-loop peak. (E) Bar graphs indicating numbers of R-loop peaks within two kb of a breakpoint junction in RDC-genes in NSPCs. (F) RPKM-normalized DRIP-seq signal over the indicated RDC-genes. Combined signal from DRIP samples and matching input controls from three biological replicates of aphidicolin-treated NSPCs is plotted. RPKM-normalized NSPC GRO-seq signal is shown to indicate transcription. RefSeq genes are shown in black. DSB junctions detected in NSPCs via HTGTS are indicated. (G) Left, zoomed-in visualization of DRIP-seq signal in RDC-genes Npas3 (top) and Magi2 (bottom), as in (F). Grey rectangles indicate regions analyzed by DRIP-qPCR. Right, DRIP-qPCR analysis using primers located in the regions shown on left in Npas3 and Magi2 [with (R1) or without (R2, negative control) R-loop peak signal]. Where indicated, samples were treated with RNase H (RH) prior to DRIP. Treatment with RNase H significantly suppressed the DRIP-qPCR signal, consistent with R-loop formation in the tested regions. DRIP-qPCR signal intensity (mean ± S.E.M) shows fold enrichment over the Snrpn negative control region. P values were determined by two-tailed, unpaired t test; ns, not significant. (H) RDC-genes with ≥ 10 R-loop peaks (n = 5) show a significantly earlier replication timing than RDC-genes with ≤ 1 R-loop peak (n = 8). Violin plots show the frequency distribution of replication timing ratios in the two subsets of RDC-genes. Median (blue line) and quartile lines (black) are shown. P value was determined by the Mann–Whitney U test.
