Fig. 2: MacroH2A1.1 protects from TOP1cc accumulation.

a Schematic for RADAR and TOP1 CAD-Seq assays. b Representative TSS profile plots for TOP1 CUT&RUN (green) and TOP1cc CAD-Seq (purple: IP, gray: input) in MDA-MB-231 cells. Y axes depict Z-normalized read counts. c TSS-associated TOP1cc turnover following CPT-induced damage in the presence or absence of macroH2A1.1. Schematic depicts CPT treatment times used to generate the damage. TOP1cc turnover (ΔTOP1cc) is defined as the ratio of prolonged damage (30’ CPT) over steady state (5’ CPT). Profile plot depicts Z-normalized ΔTOP1cc log₂ ratios for sh-RFP control (black) and macroH2A1.1 knockdown (1.1 KD, blue). A ΔTOP1cc log₂ ratio >0 reflects an accumulation of TOP1cc in response to damage. Two independent 30’ CPT and 5’ CPT TOP1 CAD-Seq replicates were combined for average ΔTOP1cc ratios, see Supplementary Fig. 2d for 30’ CPT and 5’ CPT TOP1 CAD-Seq profiles. d Comparison of TSS-proximal ΔTOP1cc ratios from (c), separated by RNA-Seq-derived gene expression quartiles; Q1: bottom 25%, Q4: top 25%. Boxplots show the distribution of mean ΔTOP1cc signal (TSS ± 250 bp) within each expression quartile, each data point represents the TSS of one gene. P values are based on two-sided Mann–Whitney U test for the indicated, pairwise comparisons between TSS quartiles. Box limits represent upper and lower quartiles, whiskers minimum to maximum and center lines represent the median. Similar results were obtained when analyzing replicate experiments independently. e TOP1 peak-associated ΔTOP1cc ratios as in (c). TOP1 peaks were separated into top (1.1^high) and bottom tertiles (1.1^low) based on macroH2A1.1 (FLAG-1.1) enrichment, see TOP1-centered FLAG-1.1 heatmap. f TOP1 RADAR assay with genomic DNA from MDA-MB-231 cells expressing the indicated shRNAs, prior to and at the indicated timepoints after CPT treatment (1 µM, 30 min). See Supplementary Fig. 2f for a quantification of two independent experiments. Source data are provided as a Source Data file.