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: SETX prevents GR(50) dipeptide from localizing exclusively to the nucleolus in the fly salivary gland. a We directed expression of GR(50)-GFP or membrane targeted mCD8-GFP, alone or in combination with normal FLAG-SETX(wt), to the salivary gland by crossing transgenic flies with flies expressing the OK6-GAL4 driver. Flies expressing SETX(wt) with mCD8-GFP exhibit localization of SETX throughout the nucleus (TOP), while flies expressing GR(50)-GFP display dense, punctate accumulation of GR(50) dipeptides in the nucleolus (MIDDLE). Co-expression of SETX(wt) and GR(50)-GFP yields localization of GR(50) dipeptides throughout the nucleoplasm, with partial SETX co-localization (BOTTOM). Though representative images are shown here, these identical findings were observed in all 50 cells examined per genotype. Scale bars = 25 µM. b We directed expression of GR(50)-GFP or membrane targeted mCD8-GFP, alone or in combination with normal SETX(wt), to the salivary gland by crossing transgenic flies with flies expressing the OK6-GAL4 driver. As shown here with monochrome imaging, flies expressing FLAG-SETX(wt) with mCD8-GFP exhibit localization of SETX throughout the nucleus (TOP), while flies expressing GR(50)-GFP display accumulation of GR(50) dipeptides in the nucleolus (MIDDLE). Co-expression of SETX(wt) and GR(50)-GFP reduces the accumulation of GR(50) dipeptides in the nucleolus and nucleus (BOTTOM). Scale bars = 50 µM