Epigenetic control of Topoisomerase 1 activity presents a cancer vulnerability

Abstract

DNA transactions introduce torsional constraints that pose an inherent risk to genome integrity. While topoisomerase 1 (TOP1) activity is essential for removing DNA supercoiling, aberrant stabilization of TOP1:DNA cleavage complexes (TOP1ccs) can result in cytotoxic DNA lesions. What protects genomic hot spots of topological stress from aberrant TOP1 activity remains unknown. Here, we identify chromatin context as an essential means to coordinate TOP1cc resolution. Through its ability to bind poly(ADP-ribose) (PAR), a protein modification required for TOP1cc repair, the histone variant macroH2A1.1 establishes a TOP1-permissive chromatin environment, while the alternatively spliced macroH2A1.2 isoform is unable to bind PAR or protect from TOP1ccs. By visualizing transcription-induced topological stress in single cells, we find that macroH2A1.1 facilitates PAR-dependent recruitment of the TOP1cc repair effector XRCC1 to protect from ssDNA damage. Impaired macroH2A1.1 splicing, a frequent cancer feature, was predictive of increased sensitivity to TOP1 poisons in a pharmaco-genomic screen in breast cancer cells, and macroH2A1.1 inactivation mirrored this effect. Consistent with this, low macroH2A1.1 expression correlated with improved survival in cancer patients treated with TOP1 inhibitors. We propose that macroH2A1 alternative splicing serves as an epigenetic modulator of TOP1-associated genome maintenance and a potential cancer vulnerability.

Figure 1.. MacoH2A1.1 defines TOP1 permissive chromatin domains.

(A) Heatmaps and profile plots for FLAG-macroH2A1.1 CUT&RUN signal centered on TSS-proximal (TSS) and TSS-distal (non-TSS) TOP1 peaks in MDA-MB-231 macroH2A1.1 KO cells reconstituted with FLAG-macroH2A1.1. Cells without FLAG-macroH2A1.1 served as negative control (no FLAG), a representative CUT&RUN experiment is shown. (B) IGV browser shots of distinct macroH2A1.1 chromatin environments associated with TOP1 domains (left) or facultative heterochromatin (HC) marked by H3K27me3 (right). (C) Jaccard indices for observed peak overlap between a feature of interest (top) and a reference feature (bottom), or randomly shuffled reference feature peaks of equal size (permuted). Values on the y-axis represent the intersection divided by the union in base pairs. (D) Western blot for the indicated proteins in nuclear lysates (input) or anti-FLAG IP samples from parental and FLAG-macroH2A1.1 knock-in 293 cells (F-1.1) in the presence or absence of CPT and the PARPi Olaparib. * ubiquitinated macroH2A1.

Figure 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) for the top quartile of expressed genes in MDA-MB-231 cells, as defined by RNA-Seq. Y-axes depict Z-normalized read counts. (C) TSS-associated TOP1cc resolution following CPT-induced damage in the presence or absence of macroH2A1.1. Profile plot depicts Z-normalized DTOP1cc ratios of damage-induced (30’ CPT) over steady state TOP1cc (5’ CPT) for sh-RFP control (black) and macroH2A1.1 knockdown (1.1 KD, blue). (D) Violin plots based on samples in (C) depicting mean DTOP1cc ratios within 500 bp of the TSS, separated based on RNA-Seq-derived gene expression quartiles; Q1: bottom 25%, Q4: top 25%. **** p < 3e-16 based on Wilcoxon rank-sum t-test, all quartiles are significantly different between 1.1 KD and control shRNA (p < 3e-16). (E) TOP1 peak-associated DTOP1cc 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 Fig. S2D for a quantification of two independent experiments.

Figure 3.. TOP1cc clearance depends on the macroH2A1.1 PAR binding domain.

(A) TOP1 RADAR assay with genomic DNA from macroH2A1.1 knockout MDA-MB-231 cells reconstituted with WT (+1.1) or G224E mutant FLAG-macroH2A1.1 (+1.1GE), prior to and at the indicated timepoints after CPT treatment, one of three representative experiments is shown. (B) Quantification of RADAR analysis in (A), depicting the percentage of remaining TOP1cc relative to 0’ after CPT treatment (n = 3), values are expressed as mean and SD. P values are based on student’s two-tailed t-test, * p < 0.05, ** p < 0.01.

Figure 4.. XRCC1 recruitment to TOP1ccs depends on macroH2A1.1.

(A) Western blot for the indicated proteins in nuclear lysates (input) or IP lysates from parental (P) and FLAG-macroH2A1.1 (F-1.1) or FLAG-macroH2A1.2 (F-1.2) knock-in 293 cells in the presence or absence of PARPi. (B) Quantification of XRCC1 foci in MCF7 WT and macroH2A1.1 KO cells in the presence or absence of CPT treatment (1 μM, 30 min), y-axis depicts foci per nucleus (n>350). Representative images are shown in Fig. S4A. (C) Western blot for the indicated proteins in macroH2A1.1 KO (1.1 KO) MCF7 cells reconstituted with empty vector (EV), FLAG-macroH2A1.1 (1.1) or FLAG-macroH2A1.1 G224E (1.1GE). (D) XRCC1 foci in cells from (C) treated with CPT (1 μM, 30 min) or DMSO (Mock). One of two independent experiments is shown, scale bar: 10 μm. Cells were quantified as in (B), n>300 nuclei per sample. A representative of two independent clonal 1.1 KO reconstitutions is shown. (E) Alkaline comet assay in WT and 1.1 KO MCF7 cells treated with 20 μM CPT or DMSO (Mock); y-axis depicts Olive tail moment (n>50), a representative of two independent experiments is shown, scale bar: 100 μm. For all violin plots, red lines reflect the median, P values are based on Mann-Whitney U test; ** p < 0.01, **** p < 0.0001, ns: not significant.

Figure 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. Representative YFP-MCP, XRCC1 and TOP1 IF images of nucleus 5 h after Dox-treatment are shown, PARGi was added for 30 min. Squares depict the MS2 site or a control region used for analyses in (B-D), scale bar: 10 μm. (B) Averaged signal intensities (n=82) for the indicated proteins at MS2 or control regions following MS2 induction as in (A). (C) 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 (A) (n=82). A representative of two independent experiments is shown; si-ctrl cells were used for analyses in (B). (D) Average TOP1 intensities and corresponding violin plots as in (C) (n>55). 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, red lines reflect the median, P values are based on Mann-Whitney U test; **** p < 0.0001, ns: not significant. Scale bars in B-D are 1 μm.

Figure 6.. macroH2A1.1 drives TOP1i resistance in cancer cells.

(A) Western blot for the indicated proteins in MDA-MB-453 and NCI60 breast cancer cell lines (bold). TNBC cell lines are italicized. (B) Drug activity levels based on NCI60 drug screen in the indicated breast cancer cell lines, arranged by increasing macroH2A1.1 expression; n=number of compounds tested per drug target. P values are based on Pearson’s Correlation Coefficient. Note that MDA-MB-453 cells were not part of the NCI60 data set. (C) Cell viability of the indicated cell lines in response to CPT treatment measured by MTT assay, macroH2A1.1^high cells are in gray, macroH2A1.1^low cells in blue. Data are presented as mean and SD (n=4); p < 0.001 based on Students two-tailed t-test, relative to MDA-MB-231 cells. (D) TOP1 RADAR assay for the indicated cell lines as in Fig. 2F. (E) Western blot in MCF7 cells expressing siRNAs against macroH2A1.1 (si-1.1), macroH2A1.2 (si-1.2) or a control sRNA (si-ctrl). (F) Clonogenic survival of cells from (E) in response to the indicated drug combinations. Survival was normalized to untreated cells for each siRNA transfection. Representative images are shown, data are presented as mean and SD (n=3). P values are based on Student’s two-tailed t-test, * p < 0.05, *** p < 0.001, ns: not significant. (G) Cell viability in the indicated cell lines in response to CPT treatment in the presence or absence of PARPi, measured by MTT after 10 days of clonogenic growth, data are presented as mean and SD (n=3).

Figure 7.. macroH2A1.1 expression is indicative of SSL resistance in cancer patients.

(A) Correlation between relative macroH2A1.1 expression and BER-related APOBEC signature scores (SBS2 and SBS13) in TCGA breast tumors with high mutation burden (SBS scores > 100). P values are based on Spearman correlation. (B) Kaplan-Meyer survival analysis of TCGA ovarian cancer patient subgroups where treatment regimens contained Topotecan (n=119) or taxol (n=821). Patients were stratified by macroH2A1.1 mRNA expression based on an isoform-specific Affymetrix microarray probe (214500_at), the bottom 25% were considered macroH2A1.1 low expressors.