Fig. 3: CDDD11-8 induces promoter-proximal pausing in MDA-MB-453 cells.

a Schematic showing RNA polymerase II (Pol II) localization and corresponding Pol II enrichment by ChIP-seq over promoters and gene bodies during normal transcriptional elongation (upper panel) and during promoter-proximal pausing (lower panel). b Average read density plots (top panels) and heatmaps (bottom panels) representing Pol II ChIP-seq data, showing increased enrichment of Pol II at gene promoters after 4 h treatment with CDDD11-8 (600 nM) in MDA-MB-453 cells. Heatmaps are broken into three regions (increased, decreased, and no change with CDDD11-8), determined by differential enrichment analysis using FDR < 0.05. Data is presented as an average of two replicates representing independent passages of cells. c Average empirical cumulative distribution function plot showing a rightward shift in Pol II pausing index at promoters after treatment with CDDD11-8 (as defined in (b)) in MDA-MB-453 cells. The pausing index was defined as a log-transformation of the ratio between the Pol II promoter density and gene body density, where promoters were defined between – 50 bp and + 300 bp of the TSS, and gene bodies defined between – 300 bp and + 3 kb of the TES. Data is presented as an average of two replicates representing independent passages of cells and analyzed using a paired two-sided Wilcoxon test (W = 39,578,209, n = 9,286, p < 0.0001). d Gene ontology analysis for Pol II ChIP-seq data at CDDD11-8 enriched promoters identified in (b), compared to promoter regions that were not differentially gained with treatment. Over-represented HALLMARK gene sets and their corresponding log-transformed FDR values are shown for gene sets which met an FDR-corrected p value (q) of <0.05. e Representative genome browser tracks showing the average Pol II ChIP-seq signal in MDA-MB-453 cells at select HALLMARK G2/M checkpoint binding sites where Pol II promoter-proximal pausing was induced by CDDD11-8. Tracks are scaled to the maximal value (as indicated numerically at the upper-right of each track) to highlight changes in Pol II enrichment over the gene body. The pausing index (PI) ratio between CDDD11-8 and vehicle-treated cells are inset into the CDDD11-8 Pol II tracks. Data is presented as an average of two replicates representing independent passages of cells. The genomic tracks for E2F2 have been horizontally scaled for ease of visualization, as they are normally oriented on the minus strand.