Fig. 5: MacroH2A1.1 promotes TOP1cc repair at sites of nascent transcription.

a Schematic of U2OS cell-based reporter for transcription-associated DNA damage repair. An MS2 repeat-containing transcript is induced by Dox and detected with YFP-MCP, PARG inhibitor (PARGi) treatment served to stabilize PAR chains. b YFP-MCP, XRCC1 and TOP1 IF images of a representative nucleus 5 h after Dox-treatment, PARGi was added for 30 min prior to analysis. Squares depict the MS2 site or a control region used for analyses in (d–f), scale bar: 10 µm. Similar results were obtained in three independent experiments. c TOP1 CAD-qPCR at the MS2 locus in the presence or absence of Dox in MS2 reporter cells expressing sh-RFP (sh-ctrl) or sh-macroH2A1.1 (sh-1.1). TSS-proximal and upstream qPCR primer pairs are shown in (a). IgG was used as a negative control. The y-axis depicts relative enrichment, normalized to TOP1cc levels in untreated sh-RFP cells, average and SEM are from three independent experiments. d Averaged signal intensities for the indicated proteins following MS2 induction as in (a), n = 82 MS2 or matched control loci. e XRCC1 intensity distributions 5 h after Dox/PARGi treatment in cells transfected with non-targeting control (si-ctrl), or macroH2A1.1 siRNA (si-1.1). For each MS2 site, mean fluorescence intensity was measured at the MS2 peak (white circle) or a corresponding control region as defined in (b) (n = 82 MS2 or matched control loci). A representative of two independent experiments is shown in (d, e); si-ctrl cells were used for analyses shown in (d). f Average TOP1 intensities and corresponding violin plots as in (e) (n > 55 MS2 loci, see source data for exact n). TOP1 signal was measured following 1 h of Dox treatment to avoid potential changes in TOP1 retention due to prolonged repair activity. For all violin plots, center lines (red) represent the median, white dotted lines depict upper and lower quartiles, p values are based on two-sided Mann-Whitney U test for the indicated, pairwise comparisons. Scale bars in (d–f) are 1 µm. Source data for (c, e, f) are provided as a Source Data file.