Figure 1
Elucidation of R-loops in neural stem/progenitor cells. (A) Top, illustration of genes with DNA breakpoints in the promoter region, defined as the two kilobase (kb) region surrounding the transcription start site (TSS). Triangles illustrate HTGTS breakpoint junctions. Bottom, box-and-whiskers plot showing fractional GC content in promoter regions of all NCBI37/mm9 RefSeq genes (n = 22,735) and actively transcribed genes with HTGTS junctions within two kb of the TSS in NSPCs (n = 2332). Whiskers show minimum and maximum values; upper and lower box edges correspond to the 25th and 75th percentile; horizontal line indicates the median. P < 0.0001, Mann–Whitney U test. (B) Visualization of reads per kilobase per million (RPKM)-normalized DRIP-seq signal in input controls and DRIP samples over the indicated genomic regions. Combined signal from nine DRIP samples and matching input controls from three biological replicates is plotted. RPKM-normalized GRO-seq signal is plotted to show transcription. Location of RefSeq genes and position of DSB junctions detected by high-throughput, genome-wide, translocation sequencing (HTGTS) of aphidicolin-treated NSPCs is shown. (C) Genomic distribution of NSPC R-loop peaks across the indicated genome annotations compared to the expected genomic distribution. (D) Transcriptional status of genes with R-loop peaks in NSPCs. Data in (C) and (D) represent mean ± S.E.M. from three independent DRIP-seq experiments performed on biological replicates. P values were determined by one-way ANOVA with Tukey’s post hoc correction for multiple comparisons. (E) Left, metaplot analysis of RPKM-normalized, raw DRIP-seq signal across active (blue, n = 15,528) and inactive (dark gray, n = 3246) genes in NSPCs reveals enrichment at TSSs, gene bodies, and transcription end sites (TESs). Right, RPKM-normalized, raw DRIP-seq signal around the TSSs of active and inactive genes in NSPCs.
