Fig 2: BRCA1/SETX Complex at the β-actin Pause Site Protects Cells from R-Loop-Driven ssDNA Breaks(A) ChIP analysis performed on the β-actin gene as in Figure 1 using γH2AX and total H2AX antibodies. Histograms represent the proportion of total H2AX phosphorylated on Ser139 (i.e., γH2AX). Average ChIP values ± SD from three independent experiments are shown.(B) LM-PCR strategy used to identify R-loop-associated ssDNA breaks on the coding strand. See Supplemental Experimental Procedures.(C) Quantitative detection of ssDNA after LM-PCR performed on the β-actin gene before and after BRCA1 or SETX knockdown and with or without ectopic RNaseH1 expression. QPCR values are average ± SD from three independent experiments. ∗p < 0.05, ∗∗p < 0.007 by one-tailed Student’s t test.See also Figure S3.

Fig 3: BRCA1 Recruitment to the β-actin Termination Pause Site Is Mediated by R-Loops(A) RNA:DNA immunoprecipitation (DIP) analyses performed in HeLa from mock-treated or BRCA1-depleted cells (BRCA1 KD) (left) and mock-treated or SETX-depleted cells (SETX KD) (right).(B) BRCA1 and SETX ChIP experiment performed in control conditions (−RNaseH1) or with RNaseH1 overexpression (+RNaseH1). Average DIP and ChIP values ± SD from three, independent experiments are shown. ∗p < 0.05.(C) BRCA1 and SETX co-IP experiments performed in HeLa cells ± RNaseH1 expression. IgG, negative control.

Fig 4: BRCA1 Interacts with SETX and Is Required for SETX Recruitment to the R-Loop-Associated Termination Pause Region of the Human β-actin Gene(A) Co-immunoprecipitation (IP) of endogenous BRCA1 and SETX in HeLa cell (top) and in BJ-hTert human fibroblast extracts (bottom), using antibodies against BRCA1 (BRCA1 #1/#2) or SETX (SETX #1/#2). IgG, negative control.(B) In vitro interaction assay using recombinant GST-BRCA1 fragments performed in HeLa cells. GSH bead-bound proteins were eluted and analyzed by immunoblot. Bottom: relative abundance of each recombinant affinity purified fusion protein (marked with a star) visualized by SDS-PAGE after Coomassie staining. Additional bands are readthrough and/or degradation products of recombinant proteins.(C) Same as in (B) but with recombinant GST-SETX fragments.(D) Schematic representation of the human β-actin gene: exons are numbered, and the red box reflects the existence of a transcription pause element. Location of primers used for the ChIP experiments are depicted above the diagram.(E) Immunoblot that reflects the efficiency of the siRNA-mediated depletion of BRCA1 and SETX. Top: SETX and BRCA1; bottom: Vinculin used as a loading control.(F) ChIP analyses performed on the β-actin gene from mock-treated (red bars), BRCA1- (green bars), or SETX-depleted cells (gray bars) using BRCA1 Ab #3. Average ChIP values ± SD from three independent experiments are shown.(G) Same ChIP experiments as in (F) but with SETX Ab #1.See also Figures S1 and S2.

Fig 5: PARP1 associates with R-loop-forming sites in cells. (A) Representative images of S9.6:PARP1 PLA in RNH1-WT-GFP and RNH1-D210N-GFP U2OS cells untreated or induced with DOX. PLA foci (red) are observed due to PARP1 antibody association within a 40 nm distance of the S9.6 antibody. White scale bars represent 10 µm. (B) Quantification of the number of PLA foci per nucleus for each experimental condition illustrated in (A). Orange bars represent the mean. Data are from three independent experiments with 75 to 100 cells counted per experiment. P values were obtained using ordinary one-way ANOVA. (C) Representative images of S9.6:PARP1 PLA analysis in U2OS cells transfected with siRNA for RNH1 or SETX. White scale bars represent 10 µm. (D) Quantification of the number of PLA foci per nucleus for each experimental condition illustrated in (C). Orange bars represent the mean. Data are collected from 100 to 170 nuclei. P values were obtained using ordinary one-way ANOVA. (E) Representative images of ant-PAR immunofluorescence in RNH1-D210N-GFP U2OS cells. PAR signal is shown in red and RNH1-D210N-GFP expression in green (middle row). DNA is shown in blue after DAPI staining. Aphidicolin (APH) (0.4 µM) was used as a positive control. White scale bars represent 10 µm. (F) Quantification of relative PAR signal intensity as shown in (E). Orange bars represent median PAR intensity. Data are from three independent experiments in which 100–150 nuclei were counted for each experiment. P values were obtained using one-way ANOVA analysis. (G) Western blot analysis of chromatin-bound protein fractions isolated from RNH1-WT-GFP and RNH1-D210N-GFP U2OS cells before and after induction with DOX and treatment with 100 nM talazoparib (PARPi) for 24 hrs. (H) Quantification of relative fold change in chromatin PARP1 signal over non-PARPi-treated conditions. Data are from three independent experiments. P values were obtained using Student's t-tests. (I) Representative images of S9.6:PARP1 PLA analysis in RNH1-D210N-GFP U2OS cells treated with PARPi. RNH1-D210N-GFP expression is shown in green and DNA in blue after DAPI staining. It is to be noted that the dimmer GFP signal is due to the pre-extraction and PLA staining processes (second and bottom row). White scale bars represent 10 µm. (J) Quantification of the number of PLA foci per nucleus for each experimental condition illustrated in (I). Orange bars represent the mean. Data are from 100 to 200 nuclei. P values were obtained using ordinary one-way ANOVA.